Monomeric cxcl121 peptide and use thereof

ABSTRACT

The present invention provides a CXCL121 peptide engineered to resist peptide-induced dimerization by maintaining steric repulsion of the chemokine helix, pharmaceutical compositions thereof, and methods of using said dimer in the treatment of cancer, inflammatory disorders, autoimmune disease, and HIV/AIDS.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/874,476 filed on Jan. 18, 2018, which is a continuation of U.S.application Ser. No. 14/736,535, filed Jun. 11, 2015 now U.S. Pat. No.9,908,923, which claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/010,655 filed on Jun. 11, 2014, each of which isincorporated by reference for all purposes.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH AND DEVELOPMENT

This invention was made with government support under grants R01AI058072(BFV), R01GM097381 (BFV), R56AI063325 (BFV), U01GM094612 (TMH), andR01GM081763 (TMH) awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates generally to a CXCL12₁ monomer, pharmaceuticalcompositions thereof, and methods of using the CXCL12₁ monomer in thetreatment of cancer, autoimmune and inflammatory disease, and HIV/AIDS.

BACKGROUND OF THE INVENTION

Chemokines are small soluble proteins that stimulate chemotactic cellmigration via activation of a G protein-coupled receptor (GPCR). Inaddition to their vital roles in inflammation, wound healing, and stemcell homing, chemokines also contribute to many pathologies includingautoimmune diseases and cancer. Interactions of the chemokine CXCL12(stromal cell-derived factor-1/SDF-1) and its receptor CXCR4 areparticularly well studied because of their participation inneurogenesis, cardiogenesis, angiogenesis, myocardialinfarction/reperfusion injury, HIV infection, and numerous carcinomasand sarcomas.

Chemokine receptor recognition and activation occurs via a two-step,two-site process. First, the CXCR4 extracellular N-terminus binds toCXCL12 (site 1). The N-terminus of CXCL12 then recognizes the receptortransmembrane domain and activates signaling (site 2). In addition toone site of O-linked glycosylation, CXCR4 possesses three tyrosineresidues (Tyr7, Tyr12, and Tyr21) in the N-terminus capable of beingO-sulfated in the Golgi apparatus. Mutational studies suggested thatsulfation of Tyr21 enhances the binding of wild type CXCL12(CXCL12_(WT), SEQ ID NO:1), but the level of sulfation at each tyrosineand their relative contributions to chemokine recognition were notquantified. Consistent with studies of the full-length CXCR4 receptorexpressed in cells, sequential sulfation of a peptide comprisingresidues 1-38 (CXCR4₁₋₃₈) enhanced the affinity for CXCL12. Full-lengthchemokine receptors CCR2b, CCR5, CCR8, CXCR3, and CX₃CR1 have since beenshown to also exhibit increased ligand binding affinity upon tyrosinesulfation; replacement of tyrosines with phenylalanine residues resultedin 10 to 200-fold decreases in affinity and, in some cases, undetectablebinding demonstrating the importance of sulfotyrosine recognition inchemokine signaling.

While most chemokines form dimers or other oligomers, receptoractivation is typically restricted to the monomeric ligand. However,structure-function studies of CXCL12 using preferentially monomeric(CXCL12_(H25R)) and constitutively dimeric (CXCL12₂) variantsdemonstrated that dimerization converts CXCL12 into a partial CXCR4agonist that potently inhibits chemotaxis. As a CXCR4 ligand thatstimulates intracellular calcium flux but fails to activate F-actinpolymerization or β-arrestin recruitment, the CXCL12₂ dimer causes atype of ‘cellular idling’ that can block metastatic tumor formation inanimal models for colorectal cancer and melanoma. Differences in how theCXCR4 N-terminus is recognized by CXCL12 monomers and dimers maycontribute to their distinct receptor activation profiles (26). Forinstance, in the NMR structure of the CXCL12₂:CXCR4₁₋₃₈ complex thereceptor fragment wraps around both subunits of the CXCL12 dimer,placing sulfotyrosine 12 (sTyr12) and sTyr21 in distinct sites on onesubunit while sTyr7 occupies a cleft at the dimer interface that wouldnot exist in a CXCL12 monomer. In concordance with this structuralmodel, it was observed that CXCR4₁₋₃₈ binding promotes CXCL12dimerization. However, individual contributions of CXCR4 sulfotyrosinesto the affinity and specificity of CXCL12 recognition and their impacton the monomer-dimer equilibrium remain unknown.

Site 1 contacts that contribute most to binding are potential targetsfor development of novel chemokine probes and antagonists. For example,we recently demonstrated that the sTyr21 binding pocket of CXCL12 can betargeted for inhibition by small molecule ligands that blockCXCR4-mediated calcium signaling and chemotaxis (29, 30). This appearsto be a conserved binding site, suggesting that sulfotyrosine-guideddrug discovery may be a general strategy for targeting the chemokinefamily and other protein-protein interactions in the extracellularspace.

Accordingly, there is a current need for cost-effective pharmaceuticalagents and treatment methods for treating various conditions includingautoimmune or inflammation disorders, immune suppression conditions,infections, blood cell deficiencies, cancers and other describedconditions and to mobilize stem cells by manipulating and controllingCXCL12 and CXCR4.

SUMMARY OF THE INVENTION

The present invention provides a CXCL12₁ peptide. In one embodiment, theCXCL12₁ peptide has been engineered to resist peptide-induceddimerization by maintaining steric repulsion of the chemokine helix.Specifically, in one embodiment, the CXCL12₁ peptide (SEQ ID NO:2)comprises L55C and I58C substitutions as compared to the wild-typepeptide (SEQ ID NO:1).

In other embodiments, the invention provides a composition comprising aCXCL12₁ peptide, wherein the peptide comprises L55C and I58Csubstitutions, and a pharmaceutically acceptable carrier or diluent.

In other embodiments, the invention provides a method of treating anautoimmune disease in a subject comprising administering to the subjecta therapeutically effective amount of a composition comprising a CXCL12₁peptide.

In other embodiments, the invention provides a method of treating asolid tumor in a subject comprising administering to the subject atherapeutically effective amount of a composition comprising a CXCL12₁peptide.

In other embodiments, the invention provides a method of inhibitingangiogenesis in a subject by administering to the subject atherapeutically effective amount of a composition comprising a CXCL12₁peptide.

In other embodiments, the invention provides a method of treating cancerin a subject comprising administering to the subject a therapeuticallyeffective amount of a composition comprising a CXCL12₁ peptide.

In other embodiments, the invention provides a method of treatingHIV/AIDS in a subject comprising administering to the subject atherapeutically effective amount of a composition comprising a CXCL12₁peptide.

In other embodiments, the invention provides a method of inducingapoptosis in a subject comprising administering to the subject atherapeutically effective amount of a composition comprising a CXCL12₁peptide.

In other embodiments, the invention provides a method of treatingleukemia in a subject comprising administering to the subject atherapeutically effective amount of a composition comprising a CXCL12₁peptide, wherein the leukemia cells express CXCR4.

In other embodiments, the invention provides a method of inhibitingmigration of cancer cells in a subject comprising administering to thesubject a therapeutically effective amount of a composition comprising aCXCL12₁ peptide, wherein the cancer cells express CXCR4.

In other embodiments, the invention provides a method of inhibitingmigration of leukemia cells in a subject comprising administering to thesubject a therapeutically effective amount of a composition comprising aCXCL12₁ peptide, wherein the leukemia cells express CXCR4. In oneembodiment, the leukemia cells are selected from the group consisting ofleukemia, lymphoma and myeloma.

In other embodiments, the invention provides a kit comprising a CXCL12₁peptide, a pharmaceutically acceptable carrier or diluent, andinstructional material.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A. CXCL12₁ and CXCL12₂ have discrete CXCR4₁₋₃₈ binding sites. TheCXCL12 NMR structure (PDB ID 1SDF) solved in acetate (pH 4.9) was usedto identify and model the I55C/L58C mutations for disulfide formation(yellow). Dimerization is inhibited when the helix is constrained to anacute angle relative to the β-sheet.

FIG. 1B. Chemical shift perturbations (orange) produced by CXCR4₁₋₃₈mapped onto CXCL12₁ (PDB ID 1SDF).

FIG. 1C. Chemical shift perturbations (orange) produced by CXCR4₁₋₃₈mapped onto CXCL12₂ (PDB ID 2K01). Chemical shift perturbations uniqueto CXCL12₁ are highlighted in ruby. Both structures are rotated 180°relative to their respective ribbon representations.

FIG. 1D. CXCR4₁₋₃₈ induced chemical shift perturbations fitted to aquadratic binding equation resulted in CXCL12₁ and CXCL12₂ affinities of3.5±0.1 and 0.9±0.3 μM, respectively.

FIG. 2A. Chemical shift perturbations of CXCR4 sulfopeptides designedfrom the extracellular N-terminus. The residues corresponding to thesTyr7 (cyan), sTyr12 (wheat), and sTyr21 (green) heptapeptides areindicated on the CXCR4 N-terminus amino acid sequence. The previouslydefined positions of sTyr7.

FIG. 2B. The residues corresponding to the sTyr7 (cyan), sTyr12 (wheat),and sTyr21 (green) beptapeptides are indicated on the CXCR4 N-terminusamino acid sequence. The previously defined positions of sTyr12.

FIG. 2C. The previously defined positions of sTyr21.

FIG. 2D. The previously defined positions of heptapeptides arereproduced from the CXCL12₂:CXCR4₁₋₃₈ NMR structure (PDB ID 2K05).

FIG. 2E Chemical shift perturbations induced by sTyr7. Chemical shiftperturbations identify distinct binding sites for the sTyr7.

FIG. 2F. Chemical shift perturbations induced by sTyr7. Chemical shiftperturbations identify distinct binding sites for the sTyr7.

FIG. 2G. Chemical shift perturbations induced by sTyr7. Chemical shiftperturbations identify distinct binding sites for the sTyr7.

FIG. 2H. Chemical shift perturbations induced by sTyr7. Chemical shiftperturbations identify distinct binding sites for the sTyr7.

FIG. 2I. Chemical shift perturbations induced by sTyr12. Non-specificbinding was observed for the sTyr12 sulfopeptide.

FIG. 2J. Chemical shift perturbations induced by sTyr2. Non-specificbinding was observed for the sTyr12 sulfopeptide.

FIG. 2K. Chemical shift perturbations induced by sTyr12. Non-specificbinding was observed for the sTyr12 sulfopeptide.

FIG. 2L. Chemical shift perturbations induced by sTyr12. Non-specificbinding was observed for the sTyr12 sulfopeptide.

FIG. 2M. Chemical shift perturbations induced by sTyr21 sulfopeptidesmap to the Tyr21 pocket on CXCL12₂ (PDB 2K05). Chemical shiftperturbations identify distinct binding sites for the sTyr21.

FIG. 2N. Chemical shift perturbations induced by sTyr21 sulfopeptidesmap to the Tyr21 pocket on CXCL12₂ (PDB 2K05). Chemical shiftperturbations identify distinct binding sites for the sTyr21.

FIG. 2O. Chemical shift perturbations induced by sTyr21 sulfopeptidesmap to the Tyr21 pocket on CXCL12₂ (PDB 2K05). Chemical shiftperturbations identify distinct binding sites for the sTyr21.

FIG. 2P. Chemical shift perturbations induced by sTyr21 sulfopeptidesmap to the Tyr21 pocket on CXCL12₂ (PDB 2K05). Chemical shiftperturbations identify distinct binding sites for the sTyr21.

FIG. 3A. Sulfation of Tyr21 improves sulfopeptide binding affinity andmodulates full-length receptor activity. The largest chemical shiftperturbations (orange) are consistent with the putative sTyr21sulfopeptide (green) binding site on CXCL12₂ (PDB ID 2K05).

FIG. 3B. Chemical shift perturbations induced by sulfated and unsulfatedpeptides were fitted to a quadratic binding equation to yield K_(d)values. The sTyr21 sulfopeptide (circles) bound CXCL12_(WT) withK_(d)=1.8±0.2 mM (black), CXCL12₁ with K_(d)=1.6±0.2 mM (blue) andCXCL12₂ with K_(d)=211±23 μM (red). The Tyr21 peptide (triangles) boundCXCL12_(WT) with K_(d)=2.7±0.5 mM (black), CXCL12₁ with K_(d)=1.5±0.4 mM(blue) and CXCL12₂ with K_(d)=831±137 μM (red).

FIG. 3C. The calcium response of FLAG-tagged CXCR4 variants was measuredas a function of CXCL12_(WT) concentration. Data are representative oftwo experiments each performed with three replicates.

FIG. 3D. Four parameter fits yielded each CXCR4 variants EC₅₀ andmaximum calcium response. CXCR4 variants with EC₅₀ or maximum calciumresponse values more than three standard deviations from the meanCXCR4_(WT) quantities are indicated with an asterisk.

FIG. 4A. The sTyr21 binding site is allosterically linked to CXCL12dimerization. Intrinsic tryptophan fluorescence was used to calculatethe CXCL12_(WT) dimerization affinity alone (circles; K_(d)=15.1±0.4mM), in the presence of 50 mM sulfotyrosine (squares; K_(d)=8.9±1.8 mM),or in the presence of 3 mM sTyr21 sulfopeptide (diamonds; K_(d)=2.6±0.4mM).

FIG. 4B. FP and NMR derived binding affinities were used to produce athermodynamic cycle, which illustrates that CXCL12 dimerization andsTyr21 sulfopeptide binding are coupled. After one ligand has bound, theaffinity for the second ligand is enhanced with a cooperativity factor.

FIG. 5A. Comparison of reducing (left three lanes) and non-reducing(right three lanes) SDS-PAGE of CXCL12₁, CXCL12_(WT) and CXCL12₂.

FIG. 5B. Chemical shifts in the ¹H-¹⁵N HSQC of CXCL12₁ are negligiblyperturbed upon dilution from 2 mM (black) to 50 μM (magenta) in 100 mMNaPO₄ (pH 7.4). In comparison CXCL12_(WT) and CXCL12_(H25R) exhibitdimerization K_(d)=140 μM and 1522 μM under identical solutionconditions.

FIG. 5C. SEC-MALS analysis of 40 mg ml⁻¹ CXCL12₁ (red) and CXCL12₂(black) resulted in homogenous, monodisperse peaks of 8711±0.1% g mol⁻¹and 17920±0.1% g mol⁻¹, respectively. The theoretical molar masses ofCXCL12₁ and CXCL12₂ are 7937 g mol⁻¹ and 15958 g mol⁻¹, respectively.

FIG. 6A. Chemical shift mapping of ₁₈SGD₅YDSM₂₄ (SEQ ID NO:4) and₁₈SGDYDSM₂₄ (SEQ ID NO:5) peptides on CXCL12 variants. Sulfated andunsulfated Tyr21 peptides produce similar chemical shift changes in theTyr21 pocket of CXCL12_(WT).

FIG. 6B. Sulfated and unsulfated Tyr21 peptides produce similar chemicalshift changes in the Tyr21 pocket of CXCL12₂

FIG. 6C. Sulfated and unsulfated Tyr21 peptides produce similar chemicalshift changes in the Tyr21 pocket of CXCL12₁

FIG. 7A. Chemical shift mapping of ₉SDN₅YTEE₁₅ (SEQ ID NO:6) and₉SDNYTEE₁₅ (SEQ ID NO:7) peptides on CXCL12 variants. No conserved Tyr12pocket was observed for any Tyr12 peptide variant on CXCL12_(WT).

FIG. 7B. No conserved Tyr12 pocket was observed for any Tyr12 peptidevariant on CXCL12₂.

FIG. 7C. No conserved Tyr12 pocket was observed for any Tyr12 peptidevariant on CXCL12₁.

FIG. 8A. The ₄ISIYTSD₁₀ (SEQ ID NO:8) peptide binds similar pockets onCXCL12_(WT) and CXCL12₁. The ₄ISIYTSD₁₀ (SEQ ID NO:8) peptide inducessignificant chemical shifts at the Tyr7 pocket of CXCL12_(WT).

FIG. 8B. The ₄ISIYTSD₁₀ (SEQ ID NO:8) peptide binds similar pockets onCXCL12_(WT) and CXCL12₁. The ₄ISIYTSD₁₀ (SEQ ID NO:8) peptide inducessignificant chemical shifts at the Tyr7 pocket of CXCL12₁.

FIG. 8C. Sulfation at Tyr7 reduces sulfopeptide affinity for CXCL12_(WT)from 1.9±0.3 mM to 4.3±0.3 mM.

FIG. 8D. Sulfation at Tyr7 reduces sulfopeptide affinity for CXCL12₁from 1.1±0.6 mM to 6.1±1.3 mM.

FIG. 9. FLAG-tagged CXCR4 variants possess similar surface expression.CHO cells transiently-transfected with FLAG-tagged CXCR4 variants werestrained with either IgG1 control or anti-FLAG and then analyzed by flowcytometry. Results are representative of two experiments with threereplicates each.

FIG. 10A. CXCL12₁ enhances CXCR4-mediated calcium flux, migration, andarrestin recruitment. The affinities of CXCL12 variants for CXCR4 weredetermined by ¹²⁵I-CXCL12 displacement. K_(d) values for binding ofCXCL12_(WT) and CXCL12₁ were calculated as 1.4±1.5 nM and 0.97±1.5 nM,respectively, from their corresponding log EC₅₀ values of −8.867±0.08and −8.459±0.06.

FIG. 10B. CXCL12₁ induced a CXCR4-mediated calcium response with anEC₅₀=7.1±1.3 nM similar to the CXCL12_(WT) EC₅₀=8.7±1.7 nM.

FIG. 10C. THP-1 cell chemotaxis was quantified after 3 h stimulation.The chemotactic index was calculated by normalizing the number of cellsthat migrated toward the stimulus to the number that migrated in theabsence of stimulus.

FIG. 10D. NALM6 cell migration was quantified after 90 min stimulation.Chemotaxis was determined from counting the number of migrated cells infive high power magnification fields.

FIG. 10E. Migration of MiaPaCa2 was monitored after 6 h stimulationusing Transwell migration chambers. Chemotaxis was determined fromcounting the number of migrated cells in five high power magnificationfields.

FIG. 10F. U-937 cells were confined to a 1 μl agarose droplet andmigration was observed following 18-24 h incubation with test mediacontaining CXCL12_(WT) or CXCL12₁. Migration inhibition presented asmean±SD with chemokine-free control normalized to zero.

FIG. 10G. HEK293 cells transiently co-expressing β-arrestin-2-RLuc as aBRET donor and CXCR4-YFP as BRET acceptor were stimulated withincreasing chemokine concentrations resulting in EC₅₀ values of 17.6±1.1nM for CXCL12_(WT) and 30.6±1.1 nM for CXCL12₁. CXCL12_(WT) and CXCL12₁responses were compared at each dose by two-tailed T-test (*, p<0.01; *,p<0.01; ***, p<0.001).

FIG. 11A. NMR structure of CXCL12₁ in complex with CXCR4₁₋₃₈. Surfacerepresentation of CXCL12₁ (gray) in complex with CXCR4₁₋₃₈ (orange). Forvisual clarification, CXCR4₁₋₃₈ tyrosine residues are represented byball and stick, and only CXCR4₁₋₃₈ residues 1-28 are displayed.Previously published CXCL12₁ chemical shift perturbations induced byCXCR4₁₋₃₈ are mapped onto the surface in blue.

FIG. 11B. NMR structure of CXCL12₁ in complex with CXCR4₁₋₃₈. Surfacerepresentation of CXCL12₁ (gray) in complex with CXCR4₁₋₃₈ (orange). Forvisual clarification, CXCR4₁₋₃₈ tyrosine residues are represented byball and stick, and only CXCR4₁₋₃₈ residues 1-28 are displayed.Previously published CXCL12₁ chemical shift perturbations induced byCXCR4₁₋₃₈ are mapped onto the surface in blue.

FIG. 11C. NMR structure of CXCL12₁ in complex with CXCR4₁₋₃₈. Surfacerepresentation of CXCL12₁ (gray) in complex with CXCR4₁₋₃₈ (orange). Forvisual clarification, CXCR4₁₋₃₈ tyrosine residues are represented byball and stick, and only CXCR4₁₋₃₈ residues 1-28 are displayed.Previously published CXCL12₁ chemical shift perturbations induced byCXCR4₁₋₃₈ are mapped onto the surface in blue.

FIG. 11D. The CXCL12₁:CXCR4₁₋₃₈ NMR structure (gray and orange) wasaligned to the CXCL12₂:CXCR4₁₋₃₈ NMR structure (yellow and blue; PDB2K04) with a backbone RMSD=1.41 Å. The position of Tyr21 Ca and C aretranslated by an average of 5 Å and 7 Å, respectively.

FIG. 11E. Representation of beta-sheet hydrogen bond network betweenCXCL12₁ (gray) and CXCR4₁₋₃₈ (orange) Hydrogen bonds are indicated withblack dashed lines.

FIG. 11F. CXCR4₁₋₃₈ residues Ile4 and Ile6 pack into a cleft againstresidues CXCL12₁ residues Leu26 and Tyr61.

FIG. 11G. CXCL12₁:CXCR4₁₋₃₈ complex NMR structures.

FIG. 11H. CXCL12₂:CXCR4₁₋₃₈ complex NMR structures.

FIG. 12A. CXCL12 tertiary structure controls CXCR4 fate and function.HeLa cells were stimulated with vehicle or 80 ng/ml CXCL12 variants for2 h. Endogenous CXCR4 receptor levels were determined by SDS-PAGEfollowed by immunoblotting with an anti-CXCR4 antibody. Bars representthe mean CXCR4 degraded±S.E.M., n=3.

FIG. 12B. HeLa cells were stimulated with vehicle or 80 ng/ml CXCL12variants for 20 min. Cells were stained with a PE-conjugated anti-CXCR4antibody or isotype control antibody, and endogenous CXCR4 surfaceexpression was analyzed by flow cytometry. Bars represent the mean CXCR4internalized±S.E.M., n=3.

FIG. 12C. HEK293 cells stably expressing HA-CXCR4 were stimulated withvehicle or 80 ng/ml CXCL12 variants for 30 min.

FIG. 12D. HA-CXCR4 was immunoprecipitated using an anti-HA polyclonalantibody and samples were analyzed by immunoblotting to detectincorporated FLAG-ubiquitin. Shown are representative blots from one ofthree independent experiments performed.

FIG. 12E. HeLa cells transiently transfected with HA-CXCR4-YFP werestimulated for 30 min with vehicle.

FIG. 12F. HeLa cells transiently transfected with HA-CXCR4-YFP werestimulated for 30 min with 80 ng/ml CXCL12_(WT),

FIG. 12G. HeLa cells transiently transfected with HA-CXCR4-YFP werestimulated for 30 min with 80 ng/ml CXCL12₁.

FIG. 12H. HeLa cells transiently transfected with HA-CXCR4-YFP werestimulated for 30 min with 80 ng/ml CXCL12₂. Cells were fixed,permeabilized and stained with an anti-CXCR4-p324/5 monoclonal antibodyand analyzed by confocal immunofluorescence microscopy. Shown in greenis YFP-tagged CXCR4 (far left panels) and shown in red is staining forphosphorylated CXCR4 Ser324 and Ser325 (middle left panels).Co-localization between YFP-tagged CXCR4 and phosphorylated CXCR4 appearyellow in the merge (middle right panels). Differential interferencecontrast (DIC) images are also shown (far right panels). Shown arerepresentative micrographs from three independent experiments. Bars, 20μm.

FIG. 12I. KG1a cells transiently expressing CXCR4-YFP were stimulatedwith increasing concentrations of each CXCL12 variant. Cells werestained with APC-conjugated annexin V and apoptosis was quantified byflow cytometry. Points denote the mean percentage of cells positive forannexin V±S.E.M, n=3. The level of annexin V staining was significantlydifferent between both CXCL12_(WT) and CXCL12₂, and CXCL12₁ and CXCL12₂,at all concentrations except 1 ng/ml (p<0.05).

FIG. 12J. To confirm apoptosis, PARP cleavage was monitored in KG1acells transiently transfected with CXCR4-YFP stimulated with 100 ng/mlCXCL12_(WT), CXCL12₁, or CXCL12₂. Bars denote the percentage of cellspositive for cleaved PARP S.E.M., n=3. (*, p<0.05; **, p<0.001).

FIG. 13. Model of CXCL12₁ complexed to full-length CXCR4 receptor.

FIG. 14A. The α-helix angle of CXCL12₁ is inconsistent withdimerization. CXCL12₁ residues 9-49 aligned to respective residues ofPDB ID 1SDF with a backbone RMSD=1.6 Å.

FIG. 14B. Two CXCL12₁ molecules (cyan and yellow) are aligned with eachprotomer of the CXCL12₂ NMR structure (PDB ID 2K05). The alignmentspossess backbone RMSD of 1.41 Å and 1.37 Å. The α-helices of the twoCXCL12₁ molecules possess and average angle of 65°, relative to theβ-sheet, and would sterically clash in this orientation.

FIG. 14C. Two CXCL12₁ molecules (cyan and yellow) are aligned with eachprotomer of the CXCL12₂ NMR structure (PDB ID 2K05) with the entire 20model ensemble visible.

FIG. 15A. CXCR4 residues 7-9 form a fourth β-strand with CXCL12₁.Secondary chemical shifts were calculated for CXCR4₁₋₃₈. The consensusof H^(N), N, C′, H^(α), C^(α) and C^(β) secondary chemical shiftsindicate a short β-sheet comprising residues 5-8.

FIG. 15B. The Ramachandran statistics for Tyr7, Thr8, and Ser9 areconsistent with a β-sheet.

FIG. 16A. CXCL12₁ and CXCL12₂ interact disparately with the CXCR4N-terminus. ¹H/¹⁵N heteronuclear NOE experiment of 250 μM[U-¹⁵N]-CXCR4₁₋₃₈ in the absence (orange) and presence (green) of 500 μMCXCL12₁. CXCR4₁₋₃₈ residues 4-7 exhibit a more stable interaction withCXCL12₁ than with CXCL12₂.

FIG. 16B. 2D ¹H/¹⁵N HSQC spectra of [U-¹⁵N]-CXCR4₁₋₃₈ in 25 mM d-MES (pH6.8) titrated with increasing concentrations (orange to green) ofCXCL12₁

FIG. 16C. 2D′H/¹⁵N HSQC spectra of [U-¹⁵N]-CXCR4₁₋₃₈ in 25 mM d-MES (pH6.8) titrated with increasing concentrations (orange to green) ofCXCL12₂).

FIG. 16D. CXCL12₁ and CXCR12₂ perturb CXCR4₁₋₃₈ distinctly. In someinstances, as illustrated with T13, the direction of their perturbationscan be concatenated to produce CXCL12_(WT) perturbation trajectories.

FIGS. 17A-C. CXCR4 Ile4 and Ile6 are critical for receptor binding andactivation.

FIG. 18A. CXCL12₁ enhances CXCR4-mediated calcium flux, migration, andarrestin recruitment. Binding of CXCL12 proteins was measured byradioligand displacement of ¹²⁵I-CXCL12 from CXCR4-containing membranefragments. K_(d) values for CXCR4 binding of CXCL12_(WT) and CXCL12₁were calculated as 1.44±1.5 nM and 0.97±1.5 nM, respectively, from theircorresponding log EC₅₀ values of −8.84±0.17 and −9.01±0.17.

FIG. 18B. Dose-dependent treatment of THP1 cells with either CXCL12₁ orCXCL12_(WT) induced CXCR4-mediated intracellular calcium response withEC₅₀ values of 7.1±1.3 and 8.7±1.7 nM, respectively.

FIG. 18C. NALM6 cell migration was quantified after 90 min stimulation.Chemotaxis was determined from counting the number of migrated cells infive high power magnification fields.

FIG. 18D. Migration of MiaPaCa2 was monitored after 6 h stimulationusing Transwell migration chambers. Chemotaxis was determined fromcounting the number of migrated cells in five high power magnificationfields.

FIG. 18E. U-937 cells were confined to a 1 μl agarose droplet andmigration was observed following 18-24 h incubation with test mediacontaining CXCL12_(WT) or CXCL12₁. Migration inhibition presented asmean±SD with chemokine-free control normalized to zero.

FIG. 18F. HEK293 cells transiently co-expressing β-arrestin-2-RLuc as aBRET donor and CXCR4-YFP as BRET acceptor were stimulated withincreasing chemokine concentrations resulting in EC₅₀ values of 17.6±1.1nM for CXCL12_(WT) and 30.6±1.1 nM for CXCL12₁. CXCL12_(WT) and CXCL12₁responses were compared at each dose by two-tailed T-test (*, p<0.01; *,p<0.01; ***, p<0.001).

FIG. 19A. CXCL12 tertiary structure controls CXCR4 fate and function.HeLa cells were stimulated with vehicle or 80 ng/ml CXCL12 variants for2 h. Endogenous CXCR4 receptor levels were determined by SDS-PAGEfollowed by immunoblotting with an anti-CXCR4 antibody. Bars representthe mean CXCR4 degraded±S.E.M., n=3.

FIG. 19B. HeLa cells were stimulated with vehicle or 80 ng/ml CXCL12variants for 20 min. Cells were stained with a PE-conjugated anti-CXCR4antibody or isotype control antibody, and endogenous CXCR4 surfaceexpression was analyzed by flow cytometry. Bars represent the mean CXCR4internalized±S.E.M., n=3.

FIG. 19C. HEK293 cells stably expressing HA-CXCR4 were stimulated withvehicle or 80 ng/ml CXCL12 variants for 30 min. HA-CXCR4 wasimmunoprecipitated using an anti-HA polyclonal antibody and samples wereanalyzed by immunoblotting to detect incorporated FLAG-ubiquitin. Shownare representative blots from one of three independent experimentsperformed.

FIG. 19D. HEK293 cells stably expressing HA-CXCR4 were stimulated withvehicle or 80 ng/ml CXCL12 variants for 30 min. HA-CXCR4 wasimmunoprecipitated using an anti-HA polyclonal antibody and samples wereanalyzed by immunoblotting to detect incorporated FLAG-ubiquitin. Shownare representative blots from one of three independent experimentsperformed.

FIG. 19E. HeLa cells transiently transfected with HA-CXCR4-YFP werestimulated for 30 min with vehicle or 80 ng/ml.

FIG. 19F. HeLa cells transiently transfected with HA-CXCR4-YFP werestimulated for 30 min with CXCL12_(WT).

FIG. 19G. HeLa cells transiently transfected with HA-CXCR4-YFP werestimulated for 30 min with CXCL12₁.

FIG. 19H. HeLa cells transiently transfected with HA-CXCR4-YFP werestimulated for 30 min with CXCL12₂. Cells were fixed, permeabilized andstained with an anti-CXCR4-p324/5 monoclonal antibody and analyzed byconfocal immunofluorescence microscopy. Shown in green is YFP-taggedCXCR4 (far left panels) and shown in red is staining for phosphorylatedCXCR4 Ser324 and Ser325 (middle left panels). Co-localization betweenYFP-tagged CXCR4 and phosphorylated CXCR4 appear yellow in the merge(middle right panels). Differential interference contrast (DIC) imagesare also shown (far right panels). Shown are representative micrographsfrom three independent experiments. Bars, 20 μm.

FIG. 19I. KG1a cells transiently expressing CXCR4-YFP were stimulatedwith increasing concentrations of each CXCL12 variant. Cells werestained with APC-conjugated annexin V and apoptosis was quantified byflow cytometry. Points denote the mean percentage of cells positive forannexin V±S.E.M, n=3. The level of annexin V staining was significantlydifferent between both CXCL12_(WT) and CXCL12₂, and CXCL12₁, andCXCL12₂, at all concentrations except 1 ng/ml (p<0.05).

FIG. 19J. To confirm apoptosis, PARP cleavage was monitored in KG1acells transiently transfected with CXCR4-YFP stimulated with 100 ng/mlCXCL12_(WT), CXCL12₁, or CXCL12₂. Bars denote the percentage of cellspositive for cleaved PARP±S.E.M., n=3. (*, p<0.05; **, p<0.001).

FIG. 20A. NMR structure of CXCL12₁ in complex with CXCR4₁₋₃₈. Surfacerepresentation of CXCL12₁ (gray) in complex with CXCR4₁₋₃₈ (orange). Forvisual clarification, CXCR4₁₋₃₈ tyrosine residues are represented byball and stick, and only CXCR4₁₋₃₈ residues 1-28 are displayed.Previously published CXCL12₁ chemical shift perturbations induced byCXCR4₁₋₃₈ are mapped onto the surface in blue.

FIG. 20B. NMR structure of CXCL12₁ in complex with CXCR4₁₋₃₈. Surfacerepresentation of CXCL12₁ (gray) in complex with CXCR4₁₋₃₈ (orange). Forvisual clarification, CXCR4₁₋₃₈ tyrosine residues are represented byball and stick, and only CXCR4₁₋₃₈ residues 1-28 are displayed.Previously published CXCL12₁ chemical shift perturbations induced byCXCR4₁₋₃₈ are mapped onto the surface in blue.

FIG. 20C. NMR structure of CXCL12₁ in complex with CXCR4₁₋₃₈. Surfacerepresentation of CXCL12₁ (gray) in complex with CXCR4₁₋₃₈ (orange). Forvisual clarification, CXCR4₁₋₃₈ tyrosine residues are represented byball and stick, and only CXCR4₁₋₃₈ residues 1-28 are displayed.Previously published CXCL12₁ chemical shift perturbations induced byCXCR4₁₋₃₈ are mapped onto the surface in blue.

FIG. 20D. The CXCL12₁:CXCR4₁₋₃₈ NMR structure (gray and orange) wasaligned to the CXCL12₂:CXCR4₁₋₃₈ NMR structure (yellow and blue; PDB2K04) with a backbone RMSD=1.41 Å. The position of Tyr21 Ca and C aretranslated by an average of 5 Å and 7 Å, respectively.

FIG. 20E. Representation of β-sheet hydrogen bond network betweenCXCL12₁ (gray) and CXCR4₁₋₃₈ (orange) Hydrogen bonds are indicated withblack dashed lines.

FIG. 20F. CXCR4₁₋₃₈ residues Ile4 and Ile6 pack into a cleft againstresidues CXCL12₁ residues Leu26 and Tyr61.

FIG. 20G. Comparison of the CXCL12₁:CXCR4₁₋₃₈ complex NMR structures.

FIG. 20H. Comparison of the CXCL12₂:CXCR4₁₋₃₈ complex NMR structures.

FIG. 20I. Binding affinity EC₅₀ of CXCL12_(WT) and CXCL12₁ to CHO-K1cells expressing CXCR4, CXCR4 (I4A/I6A), or CXCR4 (I4E/I6E).

FIG. 20J. Calcium flux EC₅₀ of CXCL12_(WT) and CXCL12₁ to CHO-K1 cellsexpressing CXCR4, CXCR4 (I4A/I6A), or CXCR4 (I4E/I6E).

FIG. 21A. Model and experimental validation of full-length CXCL12₁:CXCR4complex. Combination of the NMR structure and CXCR4 crystal structurepermitted modeling of the intact, balanced signaling complex. The CXCL12N-terminus is colored purple with additional site 2 interactshighlighted in pink. TCS data from Kofuku et al. are mapped onto CXCL12in orange.

FIG. 21B. The CXCL12 N-terminal residues sit in a pocket and contactresidues known to participate in chemokine binding.

FIG. 21C. R12 of the ‘site 1’ RFFESH (SEQ ID NO:10) motif actually sitsin the interface between sites. N33 may contribute to binding, butwouldn't be predicted by as a component of site 1 or 2.

FIG. 21D. Calcium flux confirms N33 contribution.

FIG. 22A. CXCL12₁ and CXCL12₂ interact disparately with the CXCR4N-terminus. 2D ¹H/¹⁵N HSQC spectra of [U-¹⁵N]-CXCR4₁₋₃₈ in 25 mM d-MES(pH 6.8) titrated with increasing concentrations (orange to green) ofCXCL12₁ (left panel) or CXCL12₂ (right panel). CXCL12₁ and CXCL12₂perturb CXCR4₁₋₃₈ distinctly. In some instances, as illustrated withT13, the direction of their perturbations can be concatenated to produceCXCL12_(WT) perturbation trajectories.

FIG. 22B. ¹H/¹⁵N heteronuclear NOE experiment of 250 μM[U-¹⁵N]-CXCR4₁₋₃₈ in the absence (orange) and presence (green) of 500 μMLM. CXCR4₁₋₃₈ residues 4-7 exhibit a more stable interaction withCXCL12₁ than with CXCL12₂.

FIG. 23A. CXCL12₁ is incapable of CXC-type dimerization. CXCL12₁ (blue)residues 9-49 aligned to respective residues of CXCL12_(WT) (gray; PDBID 1SDF) with a backbone RMSD=1.6 Å.

FIG. 23B. Two CXCL12₁ molecules (blue) are aligned with each protomer ofthe CXCL12₂ NMR structure (pink; PDB ID 2K05). The alignments possessbackbone RMSDs of 1.41 Å and 1.37 Å. The CXCL12₁ helices are oriented anaverage angle of 65° relative to the β-sheet making them stericallyinconsistent with dimerization.

FIG. 24A. CXCR4 residues 7-9 form a fourth β-strand with CXCL12₁.Secondary chemical shifts were calculated for CXCR4₁₋₃₈. The consensusof H^(N), N, C′, H^(α), C^(α) and C^(β) secondary chemical shiftsindicate a short β-sheet comprising residues 5-8.

FIG. 24B. The Ramachandran statistics for Tyr7, Thr8, and Ser9 areconsistent with a β-sheet.

FIG. 25. Expression levels for CXCR4 mutants in HEK293E cells. Theexpression of CXCR4 variants at the cell surface of the transfectedcells was assessed using flow cytometry. 48 h after transfection, 1×10⁶cells were collected and washed twice in cold PBS. Cells were thenstained with CXCR4-phycoerythrin (clone 12G5) or isotype-phycoerythrinantibody according to the manufacturer's recommendation and thefluorescence was measured using a FACSCalibur flow cytometer.

FIG. 26. CXCL12₁:CXCR4 model consistent with 2:2 binding stoichiometry.Two CXCL12₁ molecules can simultaneously maintain site 1 and site 2interactions without steric clash.

DETAILED DESCRIPTION OF THE INVENTION

Before the present materials and methods are described, it is understoodthat this invention is not limited to the particular methodology,protocols, materials, and reagents described, as these may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. As well, the terms “a” (or “an”),“one or more” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprising”, “including”, and“having” can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications and patentsspecifically mentioned herein are incorporated by reference for allpurposes including describing and disclosing the chemicals, cell lines,vectors, animals, instruments, statistical analysis and methodologieswhich are reported in the publications which might be used in connectionwith the invention. All references cited in this specification are to betaken as indicative of the level of skill in the art. Nothing herein isto be construed as an admission that the invention is not entitled toantedate such disclosure by virtue of prior invention.

I. THE INVENTION

The present invention provides a constitutively monomeric CXCL12 variant(CXCL12₁) engineered to resist peptide-induced dimerization bymaintaining steric repulsion of the chemokine helix. Six short CXCR4peptides, centered on Tyr7, Tyr12, or Tyr21, were synthesized to studythe contributions of individual sulfotyrosines in peptide binding andspecificity. Peptides were titrated into CXCL12_(WT), CXCL12₂ (aconsitutaively dimeric variant of CXCL12 containing L36C/A65Cmutations), or CXCL12₁ and the interaction was monitored by 2D NMR.While sulfopeptides encompassing sTyr7 and sTyr12 interactednonspecifically, an unsulfated Tyr7 peptide induced a new set ofchemical shift perturbations in the CXCL12 monomer that were alsoobserved upon binding of the intact CXCR4₁₋₃₈ N-terminal domain. Incontrast, the Tyr21 peptides bound specifically to the sTyr21recognition site in all three CXCL12 variants, but exhibited asignificantly higher affinity for CXCL12₂. The sTyr21 sulfopeptidecorrespondingly increased the CXCL12 dimerization affinity byeight-fold, revealing an allosteric coupling between the sulfotyrosinebinding site and CXCL12 dimerization.

Tyrosine sulfation is a post-translational modification that enhancesprotein-protein interactions and may identify druggable sites in theextracellular space. The G protein-coupled receptor CXCR4 is aprototypical example with three potential sulfation sites at positions7, 12 and 21. Each receptor sulfotyrosine participates in specificcontacts with its chemokine ligand in the structure of a soluble,dimeric CXCL12:CXCR4(1-38) complex, but their relative importance forCXCR4 binding and activation by the monomeric chemokine remainsundefined. NMR titrations with short sulfopeptides showed that thetyrosine motifs of CXCR4 varied widely in their contributions to CXCL12binding affinity and site specificity. Whereas the Tyr21 sulfopeptidebound the same site as in previously solved structures, the Tyr7 andTyr12 sulfopeptides interacted nonspecifically. Surprisingly, theunsulfated Tyr7 peptide occupied a hydrophobic site on the CXCL12monomer that is inaccessible in the CXCL12 dimer.

Functional analysis of CXCR4 mutants validated the relative importanceof individual CXCR4 sulfotyrosine modifications (Tyr21>Tyr12>Tyr7) forCXCL12 binding and receptor activation. Biophysical measurements alsorevealed a cooperative relationship between sulfopeptide binding at theTyr21 site and CXCL12 dimerization, the first example of allostericbehavior in a chemokine. Future ligands that occupy the sTyr21recognition site may act as both competitive inhibitors of receptorbinding and allosteric modulators of chemokine function. Together, ourdata suggests that sulfation does not ubiquitously enhance complexaffinity and that distinct patterns of tyrosine sulfation could encodeoligomer selectivity—implying another layer of regulation for chemokinesignaling.

CXCL12₁ Monomer.

In one embodiment, the invention provides a constitutively monomericCXCL12 variant, termed CXCL12₁, engineered to resist peptide-induceddimerization by maintaining steric repulsion of the chemokine helix.Specifically, the monomeric CXCL12 peptide of the present invention hasbeen modified to exhibit at least L55C and I58C substitutions (SEQ IDNO:2). Other substitutions are also contemplated by this invention.

In one embodiment, the CXCL12₁ monomer of the present inventioncomprises a substantially pure preparation. By “substantially pure” wemean a preparation in which more than 90%, e.g., 95%, 98% or 99% of thepreparation is that of the CXCL12₁ monomer.

The CXCL12₁ monomer of the present invention could also be incorporatedinto a larger protein or attached to a fusion protein that may functionto increase the half life of the monomer in vivo or be used as amechanism for time released and/or local delivery (U.S. Patent Appn. No.20060088510). In another embodiment, the invention provides an isolatedCXCL12₁ monomer as described above. By “isolated” we mean a nucleic acidsequence that is identified and separated from at least one component orcontaminant with which it is ordinarily associated. An isolated nucleicacid is present in a form or setting that is different from that inwhich it is found in nature. In contrast, non-isolated nucleic acidssuch as DNA and RNA are found in the state they exist in nature. Forexample, a given DNA sequence (e.g., a gene) is found on the host cellchromosome in proximity to neighboring genes; RNA sequences, such as aspecific mRNA sequence encoding a specific protein, are found in thecell as a mixture with numerous other mRNAs that encode a multitude ofproteins. However, an isolated nucleic acid encoding a given proteinincludes, by way of example, such nucleic acid in cells ordinarilyexpressing the given protein where the nucleic acid is in a chromosomallocation different from that of natural cells, or is otherwise flankedby a different nucleic acid sequence than that found in nature. Theisolated nucleic acid, oligonucleotide, or polynucleotide can be presentin single-stranded or double-stranded form. When an isolated nucleicacid, oligonucleotide or polynucleotide is to be utilized to express aprotein, the oligonucleotide or polynucleotide will contain at a minimumthe sense or coding strand (i.e., the oligonucleotide or polynucleotidecan be single-stranded), but can contain both the sense and anti-sensestrands (i.e., the oligonucleotide or polynucleotide can bedouble-stranded).

The CXCL12₁ monomer of the present invention can be prepared by standardtechniques known in the art. The peptide component of CXCL12 iscomposed, at least in part, of a peptide, which can be synthesized usingstandard techniques such as those described in Bodansky, M. Principlesof Peptide Synthesis, Springer Verlag, Berlin (1993) and Grant, G. A.(ed.). Synthetic Peptides: A User's Guide, W. H. Freeman and Company,New York (1992). Automated peptide synthesizers are commerciallyavailable (e.g., Advanced ChemTech Model 396; Milligen/Biosearch 9600).Additionally, one or more modulating groups can be attached to theCXCL12 derived peptidic component by standard methods, such as by usingmethods for reaction through an amino group (e.g., the alpha-amino groupat the amino-terminus of a peptide), a carboxyl group (e.g., at thecarboxy terminus of a peptide), a hydroxyl group (e.g., on a tyrosine,serine or threonine residue) or other suitable reactive group on anamino acid side chain (see e.g., Greene, T. W. and Wuts, P. G. M.Protective Groups in Organic Synthesis, John Wiley and Sons, Inc., NewYork (1991)). Exemplary syntheses of preferred CXCL12₁ monomer accordingto the present invention are described further in the Examples below.

Peptides of the invention may be chemically synthesized using standardtechniques such as those described in Bodansky, M. Principles of PeptideSynthesis, Springer Verlag, Berlin (1993) and Grant, G. A. (ed.).Synthetic Peptides: A User's Guide, W. H. Freeman and Company, New York,(1992) (all of which are incorporated herein by reference).

In another aspect of the invention, peptides may be prepared accordingto standard recombinant DNA techniques using a nucleic acid moleculeencoding the peptide. A nucleotide sequence encoding the peptide can bedetermined using the genetic code and an oligonucleotide molecule havingthis nucleotide sequence can be synthesized by standard DNA synthesismethods (e.g., using an automated DNA synthesizer). Alternatively, a DNAmolecule encoding a peptide compound can be derived from the naturalprecursor protein gene or cDNA (e.g., using the polymerase chainreaction (PCR) and/or restriction enzyme digestion) according tostandard molecular biology techniques.

CXCL12₁ Pharmaceutical Compositions.

In another embodiment, the invention provides a composition comprising asubstantially pure CXCL12₁ monomer of the present invention, and apharmaceutically acceptable carrier. By “pharmaceutically acceptablecarrier” we mean any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible. In oneembodiment, the carrier may be suitable for parenteral administration.Alternatively, the carrier can be suitable for intravenous,intraperitoneal, intramuscular, sublingual or oral administration.Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, membrane nanoparticle or otherordered structure suitable to high drug concentration. The carrier canbe a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. In many cases, itwill be preferable to include isotonic agents, for example, sugars,polyalcohols such as mannitol, sorbitol, or sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, such as, monostearate salts and gelatin.

Moreover, the CXCL12₁ monomer of the present invention can beadministered in a time-release formulation, such as in a compositionwhich includes a slow release polymer. The active compounds can beprepared with carriers that will protect the compound against rapidrelease, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, polylactic acid andpolylactic, polyglycolic copolymers (PLG). Many methods for thepreparation of such formulations are patented or generally known tothose skilled in the art.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g. CXCR4 agonist) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclewhich contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and freeze-drying which yieldsa powder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof. The CXCL12₁ monomerof the present invention also may be formulated with one or moreadditional compounds that enhance the solubility of the CXCL12₁ monomer.

Administration.

The CXCL12₁ monomer of the present invention, optionally comprisingother pharmaceutically active compounds, can be administered to apatient orally, rectally, parenterally, (e.g., intravenously,intramuscularly, or subcutaneously) intracisternally, intravaginally,intraperitoneally, intravesically, locally (for example, powders,ointments or drops), or as a buccal or nasal spray. Other contemplatedformulations include projected nanoparticles, liposomal preparations,resealed erythrocytes containing the active ingredient, andimmunologically-based formulations.

Parenteral administration of a pharmaceutical composition includes anyroute of administration characterized by physical breaching of a tissueof a human and administration of the pharmaceutical composition throughthe breach in the tissue. Parenteral administration thus includesadministration of a pharmaceutical composition by injection of thecomposition, by application of the composition through a surgicalincision, by application of the composition through a tissue-penetratingnon-surgical wound, and the like. In particular, parenteraladministration includes subcutaneous, intraperitoneal, intravenous,intraarterial, intramuscular, or intrasternal injection and intravenous,intraarterial, or kidney dialytic infusion techniques.

Compositions suitable for parenteral injection comprise the CXCL12₁monomer of the invention combined with a pharmaceutically acceptablecarrier such as physiologically acceptable sterile aqueous or nonaqueoussolutions, dispersions, suspensions, or emulsions, or may comprisesterile powders for reconstitution into sterile injectable solutions ordispersions. Examples of suitable aqueous and nonaqueous carriers,diluents, solvents, or vehicles include water, isotonic saline, ethanol,polyols (e.g., propylene glycol, polyethylene glycol, glycerol, and thelike), suitable mixtures thereof, triglycerides, including vegetableoils such as olive oil, or injectable organic esters such as ethyloleate. Proper fluidity can be maintained, for example, by the use of acoating such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and/or by the use of surfactants. Suchformulations can be prepared, packaged, or sold in a form suitable forbolus administration or for continuous administration. Injectableformulations can be prepared, packaged, or sold in unit dosage form,such as in ampules, in multi-dose containers containing a preservative,or in single-use devices for auto-injection or injection by a medicalpractitioner.

Formulations for parenteral administration include suspensions,solutions, emulsions in oily or aqueous vehicles, pastes, andimplantable sustained-release or biodegradable formulations. Suchformulations can further comprise one or more additional ingredientsincluding suspending, stabilizing, or dispersing agents. In oneembodiment of a formulation for parenteral administration, the CXCL12₁monomer is provided in dry (i.e., powder or granular) form forreconstitution with a suitable vehicle (e.g., sterile pyrogen-freewater) prior to parenteral administration of the reconstitutedcomposition.

The pharmaceutical compositions can be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution can be formulated according to the knownart. Such sterile injectable formulations can be prepared using anon-toxic parenterally-acceptable diluent or solvent, such as water or1,3-butanediol, for example. Other acceptable diluents and solventsinclude Ringer's solution, isotonic sodium chloride solution, and fixedoils such as synthetic mono- or di-glycerides. Otherparentally-administrable formulations which are useful include thosewhich comprise the active ingredient in microcrystalline form, in aliposomal preparation, or as a component of biodegradable polymersystems. Compositions for sustained release or implantation can comprisepharmaceutically acceptable polymeric or hydrophobic materials such asan emulsion, an ion exchange resin, a sparingly soluble polymer, or asparingly soluble salt.

The CXCL12₁ monomer of the present invention may also contain adjuvantssuch as suspending, preserving, wetting, emulsifying, and/or dispersingagents, including, for example, parabens, chlorobutanol, phenol, sorbicacid, and the like. It may also be desirable to include isotonic agents,for example, sugars, sodium chloride, and the like. Prolonged absorptionof injectable pharmaceutical compositions can be brought about by theuse of agents capable of delaying absorption, such as aluminummonostearate and/or gelatin.

Dosage forms can include solid or injectable implants or depots. Inpreferred embodiments, the implant comprises an effective amount of theCXCL12₁ monomer and a biodegradable polymer. In preferred embodiments, asuitable biodegradable polymer can be selected from the group consistingof a polyaspartate, polyglutamate, poly(L-lactide), a poly(D,L-lactide),a poly(lactide-co-glycolide), a poly(ε-caprolactone), a polyanhydride, apoly(beta-hydroxy butyrate), a poly(ortho ester) and a polyphosphazene.In other embodiments, the implant comprises an effective amount of theCXCL12₁ monomer and a silastic polymer. The implant provides the releaseof an effective amount of CXCL12₁ monomer for an extended period rangingfrom about one week to several years.

Solid dosage forms for oral administration include capsules, tablets,powders, and granules. In such solid dosage forms, the CXCL12₁ monomeris admixed with at least one inert customary excipient (or carrier) suchas sodium citrate or dicalcium phosphate or (a) fillers or extenders, asfor example, starches, lactose, sucrose, mannitol, or silicic acid; (b)binders, as for example, carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidone, sucrose, or acacia; (c) humectants, as forexample, glycerol; (d) disintegrating agents, as for example, agar-agar,calcium carbonate, potato or tapioca starch, alginic acid, certaincomplex silicates, or sodium carbonate; (e) solution retarders, as forexample, paraffin; (f) absorption accelerators, as for example,quaternary ammonium compounds; (g) wetting agents, as for example, cetylalcohol or glycerol monostearate; (h) adsorbents, as for example, kaolinor bentonite; and/or (i) lubricants, as for example, talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, or mixtures thereof. In the case of capsules and tablets, thedosage forms may also comprise buffering agents.

A tablet comprising the active ingredient can, for example, be made bycompressing or molding the active ingredient, optionally with one ormore additional ingredients. Compressed tablets can be prepared bycompressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets can be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture.

Tablets may be manufactured with pharmaceutically acceptable excipientssuch as inert diluents, granulating and disintegrating agents, bindingagents, and lubricating agents. Known dispersing agents include potatostarch and sodium starch glycolate. Known surface active agents includesodium lauryl sulfate. Known diluents include calcium carbonate, sodiumcarbonate, lactose, microcrystalline cellulose, calcium phosphate,calcium hydrogen phosphate, and sodium phosphate. Known granulating anddisintegrating agents include corn starch and alginic acid. Knownbinding agents include gelatin, acacia, pre-gelatinized maize starch,polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Knownlubricating agents include magnesium stearate, stearic acid, silica, andtalc.

Tablets can be non-coated or coated using known methods to achievedelayed disintegration in the gastrointestinal tract of a human, therebyproviding sustained release and absorption of the active ingredient. Byway of example, a material such as glyceryl monostearate or glyceryldistearate can be used to coat tablets. Further by way of example,tablets can be coated using methods described in U.S. Pat. Nos.4,256,108; 4,160,452; and 4,265,874 to form osmotically-controlledrelease tablets. Tablets can further comprise a sweetening agent, aflavoring agent, a coloring agent, a preservative, or some combinationof these in order to provide pharmaceutically elegant and palatablepreparation.

Solid dosage forms such as tablets, dragees, capsules, and granules canbe prepared with coatings or shells, such as enteric coatings and otherswell known in the art. They may also contain opacifying agents, and canalso be of such composition that they release the active compound orcompounds in a delayed manner. Examples of embedding compositions thatcan be used are polymeric substances and waxes. The active compounds canalso be in micro-encapsulated form, if appropriate, with one or more ofthe above-mentioned excipients.

Solid compositions of a similar type may also be used as fillers in softor hard filled gelatin capsules using such excipients as lactose or milksugar, as well as high molecular weight polyethylene glycols, and thelike. Hard capsules comprising the active ingredient can be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and can further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin. Soft gelatincapsules comprising the active ingredient can be made using aphysiologically degradable composition, such as gelatin. Such softcapsules comprise the active ingredient, which can be mixed with wateror an oil medium such as peanut oil, liquid paraffin, or olive oil.

Dose Requirements.

In particular embodiments, a preferred range for therapeutically orprophylactically effective amounts of CXCL12₁ may include 0.1 nM-0.1M,0.1 nM-0.05M, 0.05 nM-15 μM or 0.01 nM-10 μM. It is to be noted thatdosage values may vary with the severity of the condition to bealleviated, especially with multiple sclerosis. It is to be furtherunderstood that for any particular subject, specific dosage regimensshould be adjusted over time according to the individual need and theprofessional judgment of the person administering or supervising theadministration of the compositions, and that dosage ranges set forthherein are exemplary only and are not intended to limit the scope orpractice of the claimed composition.

The amount of CXCL12₁ monomer in the composition may vary according tofactors such as the disease state, age, sex, and weight of theindividual. Dosage regimens may be adjusted to provide the optimumtherapeutic response. For example, a single bolus may be administered,several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It is especially advantageous to formulateparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such as active compoundfor the treatment of sensitivity in individuals.

Methods of Use.

The invention also provides corresponding methods of use, includingmethods of medical treatment, in which a therapeutically effective doseof CXCL12₁ is administered in a pharmacologically acceptableformulation. Accordingly, the invention also provides therapeuticcompositions comprising the CXCL12₁ and a pharmacologically acceptableexcipient or carrier, as described above. The therapeutic compositionmay advantageously be soluble in an aqueous solution at aphysiologically acceptable pH.

In one embodiment, the invention provides a method of treatingautoimmune disease in a subject comprising administering to the subjecta therapeutically effective amount of a composition comprising CXCL12₁.By “autoimmune disease” we mean illnesses generally understood to becaused by the over-production of cytokines, lymphotoxins and antibodiesby white blood cells, including in particular T-cells. Such autoimmunediseases include but are not limited to Multiple Sclerosis (MS),Guillain-Barre Syndrome, Amyotrophic Lateral Sclerosis, Parkinson'sdisease, Alzheimer's disease, Diabetes Type I, gout, lupus, and anyother human illness that T-cells play a major role in, such as tissuegraft rejection. In addition, diseases involving the degradation ofextra-cellular matrix include, but are not limited to, psoriaticarthritis, juvenile arthritis, early arthritis, reactive arthritis,osteoarthritis, ankylosing spondylitis. osteoporosis, muscular skeletaldiseases like tendonitis and periodontal disease, cancer metastasis,airway diseases (COPD, asthma or other reactive airways disease), renaland liver fibrosis, cardio-vascular diseases like atherosclerosis andheart failure, and neurological diseases like neuroinflammation andmultiple sclerosis. Diseases involving primarily joint degenerationinclude, but are not limited to, rheumatoid arthritis, psoriaticarthritis, juvenile arthritis, early arthritis, reactive arthritis,osteoarthritis, ankylosing spondylitis. Diseases involving the eyeinclude, but are not limited to autoimmune uveitis anduveoconjunctivitis and dry eye syndrome. Diseases involvingpost-infections complications of viral or bacterial diseases such asglomerulonephritis, vasculitis, meningoencephalitis. Diseases involvingthe gastrointestinal system include but are not limited to inflammatorybowel diseases.

By “subject” we mean mammals and non-mammals. “Mammals” means any memberof the class Mammalia including, but not limited to, humans, non-humanprimates such as chimpanzees and other apes and monkey species; farmanimals such as cattle, horses, sheep, goats, and swine; domesticanimals such as rabbits, dogs, and cats; laboratory animals includingrodents, such as rats, mice, and guinea pigs; and the like. Examples ofnon-mammals include, but are not limited to, birds, fish and the like.The term “subject” does not denote a particular age or sex.

By “treating” we mean the management and care of a subject for thepurpose of combating the disease, condition, or disorder. The termsembrace both preventative, i.e., prophylactic, and palliativetreatments. Treating includes the administration of a compound of thepresent invention to prevent, ameliorate and/or improve the onset of thesymptoms or complications, alleviating the symptoms or complications, oreliminating the disease, condition, or disorder.

By “ameliorate”, “amelioration”, “improvement” or the like we mean adetectable improvement or a detectable change consistent withimprovement occurs in a subject or in at least a minority of subjects,e.g., in at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%,70%, 75%, 80%, 85%, 90%, 95%, 98%, 100% or in a range about between anytwo of these values. Such improvement or change may be observed intreated subjects as compared to subjects not treated with the CXCL12₁monomer of the present invention, where the untreated subjects have, orare subject to developing, the same or similar disease, condition,symptom or the like. Amelioration of a disease, condition, symptom orassay parameter may be determined subjectively or objectively, e.g.,self assessment by a subject(s), by a clinician's assessment or byconducting an appropriate assay or measurement, including, e.g., aquality of life assessment, a slowed progression of a disease(s) orcondition(s), a reduced severity of a disease(s) or condition(s), or asuitable assay(s) for the level or activity(ies) of a biomolecule(s),cell(s) or by detection of cell migration within a subject. Ameliorationmay be transient, prolonged or permanent or it may be variable atrelevant times during or after the CXCL12₁ monomer of the presentinvention is administered to a subject or is used in an assay or othermethod described herein or a cited reference, e.g., within about 1 hourof the administration or use of the CXCL12₁ monomer of the presentinvention to about 3, 6, 9 months or more after a subject(s) hasreceived the CXCL12₁ monomer of the present invention.

By “modulation” of, e.g., a symptom, level or biological activity of amolecule, replication of a pathogen, cellular response, cellularactivity or the like means that the cell level or activity is detectablyincreased or decreased. Such increase or decrease may be observed intreated subjects as compared to subjects not treated with the CXCL12₁monomer of the present invention, where the untreated subjects have, orare subject to developing, the same or similar disease, condition,symptom or the like. Such increases or decreases may be at least about2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%,95%, 98%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 1000% or more orabout within any range about between any two of these values. Modulationmay be determined subjectively or objectively, e.g., by the subject'sself assessment, by a clinician's assessment or by conducting anappropriate assay or measurement, including, e.g., quality of lifeassessments or suitable assays for the level or activity of molecules,cells or cell migration within a subject. Modulation may be transient,prolonged or permanent or it may be variable at relevant times during orafter the CXCL12₁ monomer of the present invention is administered to asubject or is used in an assay or other method described herein or acited reference, e.g., within about 1 hour of the administration or useof the CXCL12₁ monomer of the present invention to about 3, 6, 9 monthsor more after a subject(s) has received the CXCL12₁ monomer of thepresent invention.

By “administering” we mean any means for introducing the CXCL12₁ monomerof the present invention into the body, preferably into the systemiccirculation. Examples include but are not limited to oral, buccal,sublingual, puCXCL121onary, transdermal, transmucosal, as well assubcutaneous, intraperitoneal, intravenous, and intramuscular injection.

By “therapeutically effective amount” we mean an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result, such as reduction or reversal of angiogenesis in thecase of cancers, or reduction or inhibition of T-cells in autoimmunediseases. A therapeutically effective amount of CXCL12₁ may varyaccording to factors such as the disease state, age, sex, and weight ofthe subject, and the ability of CXCL12₁ to elicit a desired response inthe subject. Dosage regimens may be adjusted to provide the optimumtherapeutic response. A therapeutically effective amount is also one inwhich any toxic or detrimental effects of CXCL12₁ are outweighed by thetherapeutically beneficial effects.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result, such as preventing or inhibiting the rate ofmetastasis of a tumor or the onset of bouts or episodes of multiplesclerosis. A prophylactically effective amount can be determined asdescribed above for the therapeutically effective amount. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

In another embodiment, the invention provides a method of treating atumor in a subject comprising administering to the subject atherapeutically effective amount of a composition comprising the CXCL12₁monomer. By “tumor” we mean any abnormal proliferation of tissues,including solid and non-solid tumors. For instance, the composition andmethods of the present invention can be utilized to treat cancers thatmanifest solid tumors such as breast cancer, colon cancer, lung cancer,thyroid cancer, ovarian cancer and the like. The composition and methodsof the present invention can also be utilized to treat non-solid tumorcancers such as non-Hodgkin's lymphoma, leukemia and the like.

In another embodiment, the present invention provides a method ofinhibiting angiogenesis in a subject by administering to the subject atherapeutically effective amount of a composition comprising CXCL12₁. By“angiogenesis” we mean the process whereby new blood vessels penetratetissue thus supplying oxygen and nutrients while removing waste invarious pathological conditions including but not limited to diabeticretinopathy, macular degeneration, rheumatoid arthritis, inflammatorybowel disease, cancer, psoriasis, osteoarthritis, ulcerative colitis,Crohn's disease and coronary thrombosis.

In another embodiment, the present invention provides a method oftreating inflammation in a subject by administering to the subject atherapeutically effective amount of a composition comprising CXCL12₁. By“inflammation” we mean the complex biological response of vasculartissues to harmful stimuli, such as pathogens, damaged cells, orirritants. It is a protective attempt by the organism to remove theinjurious stimuli as well as initiate the healing process for thetissue. For instance, the composition and methods of the presentinvention can be utilized to treat inflammation associated with: anallergic disease such as asthma, hives, urticaria, pollen allergy, dustmite allergy, venom allergy, cosmetics allergy, latex allergy, chemicalallergy, drug allergy, insect bite allergy, animal dander allergy,stinging plant allergy, poison ivy allergy and food allergy; aneurodegenerative disease; a cardiovascular disease; a gastrointestinaldisease; a tumor such as a malignant tumor, a benign tumor, a solidtumor, a metastatic tumor and a non-solid tumor; septic shock;anaphylactic shock; toxic shock syndrome; cachexia; necrosis; gangrene;a prosthetic implant such as a breast implant, a silicone implant, adental implant, a penile implant, a cardiac implant, an artificialjoint, a bone fracture repair device, a bone replacement implant, a drugdelivery implant, a catheter, a pacemaker and a respirator tube;menstruation; an ulcer such as a skin ulcer, a bed sore, a gastriculcer, a peptic ulcer, a buccal ulcer, a nasopharyngeal ulcer, anesophageal ulcer, a duodenal ulcer and a gastrointestinal ulcer; aninjury such as an abrasion, a bruise, a cut, a puncture wound, alaceration, an impact wound, a concussion, a contusion, a thermal burn,frostbite, a chemical burn, a sunburn, a desiccation, a radiation burn,a radioactivity burn, smoke inhalation, a torn muscle, a pulled muscle,a torn tendon, a pulled tendon, a pulled ligament, a torn ligament, ahyperextension, a torn cartilage, a bone fracture, a pinched nerve and agunshot wound; a musculo-skeletal inflammation such as a muscleinflammation, myositis, a tendon inflammation, tendinitis, a ligamentinflammation, a cartilage inflammation, a joint inflammation, a synovialinflammation, carpal tunnel syndrome and a bone inflammation.

Kits.

In another embodiment, the present invention provides a kit comprising apharmaceutical composition according to the present invention andinstructional material. By “instructional material” we mean apublication, a recording, a diagram, or any other medium of expressionwhich is used to communicate the usefulness of the pharmaceuticalcomposition of the invention for one of the purposes set forth herein ina human. The instructional material can also, for example, describe anappropriate dose of the pharmaceutical composition of the invention. Theinstructional material of the kit of the invention can, for example, beaffixed to a container which contains a pharmaceutical composition ofthe invention or be shipped together with a container which contains thepharmaceutical composition. Alternatively, the instructional materialcan be shipped separately from the container with the intention that theinstructional material and the pharmaceutical composition be usedcooperatively by the recipient.

The monomer of the present invention may also further comprise adelivery device for delivering the composition to a subject. By way ofexample, the delivery device can be a squeezable spray bottle, ametered-dose spray bottle, an aerosol spray device, an atomizer, a drypowder delivery device, a self-propelling solvent/powder-dispensingdevice, a syringe, a needle, a tampon, or a dosage-measuring container.It may be desirable to provide a memory aid on the kit, e.g., in theform of numbers next to the tablets or capsules whereby the numberscorrespond with the days of the regimen that the tablets or capsules sospecified should be ingested. Another example of such a memory aid is acalendar printed on the card, e.g., as follows “First Week, Monday,Tuesday, . . . etc. . . . Second Week, Monday, Tuesday,” etc. Othervariations of memory aids will be readily apparent. A “daily dose” canbe a single tablet or capsule or several pills or capsules to be takenon a given day.

The delivery device may comprise a dispenser designed to dispense thedaily doses one at a time in the order of their intended use isprovided. Preferably, the dispenser is equipped with a memory aid, so asto further facilitate compliance with the dosage regimen. An example ofsuch a memory aid is a mechanical counter, which indicates the number ofdaily doses that have been dispensed. Another example of such a memoryaid is a battery-powered micro-chip memory coupled with a liquid crystalreadout, or audible reminder signal which, for example, reads out thedate that the last daily dose has been taken and/or reminds one when thenext dose is to be taken.

II. THE EXAMPLES

The following examples are, of course, offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way. Indeed, various modifications of the invention in additionto those shown and described herein will become apparent to thoseskilled in the art from the foregoing description and the followingexamples and fall within the scope of the appended claims.

Example 1: Engineering a CXCL12 Constitutive Monomer

The monomer-dimer equilibrium complicates the structural analysis ofCXCL12 interactions. A H25R substitution at the dimer interfacediscourages but does not prevent CXCL12 self-association, as binding ofthe CXCR4 N-terminus induces CXCL12_(H25R) dimerization. In addition toCoulomb repulsion, a steric clash between the C-terminal α-helices ofinteracting CXCL12 monomers also limits dimer formation. The originalNMR structure of CXCL12_(WT) in acetate buffer (PDB ID 1SDF) possesses ahelix orientation (˜55° relative to the β-sheet) incompatible withdimerization. After analyzing the proximity and geometry of backbone andC^(β) atoms using the program Disulfide by Design, we constructed adisulfide-constrained CXCL12 monomer using L55C and I58C substitutionsto limit helix rearrangement and prevent dimer formation (FIG. 1A).Non-reducing SDS-PAGE, 2D NMR and SEC-MALS analyses of CXCL12₁demonstrate a properly folded, monomeric species (FIG. 5). WhereasCXCL12_(WT) self-associates with a K_(d)=140 μM in 100 mM sodiumphosphate, an HSQC dilution series of CXCL12₁ in the same conditionsproduced minimal chemical shift perturbations (FIG. 5B). We concludedthat the L55C/I58C disulfide stabilizes a monomeric CXCL12 conformationthat is incompatible with dimer formation. In other examples, L66C,I28C, L62C and I38C were also tested.

The CXCL12 monomer and dimer possess distinct CXCR4₁₋₃₈ binding sites.To establish a point of reference for subsequent measurements withsmaller peptides, we monitored the binding of CXCR4₁₋₃₈ to the CXCL12₁and CXCL12₂ variants by 2D NMR. Chemical shift mapping onto the CXCL12₁model and CXCL12₂ NMR structure (FIG. 1B, 1C) highlighted perturbationsacross the β2 and β3 strands, consistent with the previous studies, butCXCL12₁ also displayed additional contacts involving residues 58-61, 65,and 66 of the helix. Nonlinear fitting of chemical shift perturbationsyielded K_(d) values of 3.5±0.1 μM (CXCL12₁) and 0.9±0.3 μM (CXCL12₂)(FIG. 1D). We recently demonstrated that perturbations induced byCXCL12_(WT) binding to [U-¹⁵N]-CXCR4₁₋₃₈ arise from the combination ofdistinct monomeric and dimeric interactions; in particular, CXCR4residues 4-9 interact strongly with the preferentially monomericCXCL12_(H25R) variant but weakly with CXCL12₂. Taken together, theseresults suggest that the CXCR4 N-terminus makes unique contacts with thehelix of monomeric CXCL12 that are occluded by dimerization of thechemokine.

CXCL12 binding of short CXCR4 sulfopeptides. Sulfation of the CXCR4N-terminal extracellular domain at either Tyr21 alone or in combinationwith Tyr12 and Tyr7 enhances its affinity for CXCL12. To assess therespective contribution of each sulfotyrosine, we synthesized shortsulfated and unsulfated CXCR4 heptapeptides centered on each of thethree tyrosine residues (FIG. 2A). Each peptide was acetylated andamidated to create uncharged N- and C-termini, respectively. A total ofsix peptides were titrated into each CXCL12 variant and monitored by 2D¹H-¹⁵N HSQC experiments. For each titration, the chemical shift changeof every residue was used to identify the binding site and calculate theinteraction affinity. Table 1 contains a complete list of peptidebinding affinities and binding energies.

TABLE 1 Equilibrium dissociation constants and Gibbs' free energy ofbinding for peptide: CXCL12 variant complexes. CXCL12_(WT) CXCL12₁CXCL12₂ ΔG ΔG ΔG Peptide K_(d) (kcal mol⁻¹) K_(d) (kcal mol⁻¹) K_(d)(kcal mol⁻¹) CXCR4₁₋₃₈  4.5 ± 2.2 μM* −7.29* 3.5 ± 0.1 μM  −7.43   0.9 ±0.3 μM −8.24 ₁₈SGDsYDSM₂₄ 1.8 ± 0.2 mM −3.74 1.6 ± 0.2 mM −3.81  211 ±23 μM −5.01 (SEQ ID NO: 4) ₁₈SGDYDSM₂₄ 2.7 ± 0.5 mM −3.50 1.5 ± 0.4 mM−3.85   831 ± 137 μM −4.20 (SEQ ID NO: 5) ₉SDNsYTEE₁₅ 1.0 ± 0.1 mM −4.091.8 ± 0.3 mM −3.74 **266 ± 38 μM −4.87 (SEQ ID NO: 6) ₉SDNYTEE₁₅ 2.1 ±0.3 mM −3.65 2.7 ± 0.8 mM −3.50  332 ± 91 μM −4.74 (SEQ ID NO: 7)₄ISIsYTSD₁₀ 4.3 ± 0.3 mM −3.22 6.1 ± 1.3 mM −3.02 **386 ± 91 μM −4.65(SEQ ID NO: 9) ₄ISIYTSD₁₀ 1.9 ± 0.3 mM −3.71 1.1 ± 0.6 mM −4.03 **418 ±60 μM −4.60 (SEQ ID NO: 8) ΔG = −RT In K_(d) @ 298 K *K_(d) and ΔGcalculated from Veldkamp et al. (32) **Non-specific binding to sY21position

As expected, the affinity of short CXCR4 peptides for all CXCL12variants was greatly reduced relative to the intact CXCR4₁₋₃₈ N-terminaldomain, with K_(d) values ranging from ˜0.2-6 mM (Table 1).Interestingly, nearly all peptides bound most tightly to the CXCL12₂locked dimer, whereas binding to CXCL12_(WT) and CXCL12₁ were roughlyequivalent. In titrations with each of the CXCL12 variants, chemicalshift mapping indicated that peptides encompassing Tyr21 consistentlybound to the previously defined sTyr21 pocket, as detailed below (FIG.2D; FIG. 6). In contrast, the Tyr7 and Tyr12 peptides did not localizeto their corresponding interaction surfaces observed in the dimericCXCL12₂:CXCR4₁₋₃₈ complex structure (FIGS. 2B, 2C). For example, in theNMR structure residues 4-10 of CXCR4 occupied a cleft at the dimerinterface enabling sTyr7 to contact the R20 and V23 side chains ofCXCL12. Here, the greatest shift perturbations (V49, T31 and F14)induced by the sTyr7 and sTyr12 sulfopeptides were more consistent withbinding to the sTyr21 recognition site (FIGS. 2, 6 and 7).

Sulfopeptides bound with higher affinity than their unsulfated analogs,as observed in previous studies with one notable exception. Binding ofthe unsulfated Tyr7 peptide to CXCL12₁ was roughly five-fold strongerthan the sTyr7 sulfopeptide (Table 1). In contrast to the sTyr7sulfopeptide, the unsulfated version induced substantial chemical shiftchanges in residues 23-25, 58-59, and 61 (FIG. 2E), matching themonomer-specific shift perturbations detected with CXCR4₁₋₃₈ (FIG. 1B).Titrations with CXCL12_(WT) produced a similar pattern of shiftperturbations and a two-fold higher affinity for the unsulfated Tyr7peptide (FIG. 8), but there was no preferential binding to the CXCL12₂dimer. Unlike Tyr21 which is rapidly modified by recombinanttyrosylprotein sulfotransferase-1 (TPST-1), Tyr7 sulfation is slow andnot consistently predicted by the available algorithms. Contacts withthe CXCR4 N-terminus are predominantly hydrophobic and favored when Tyr7is unmodified and CXCL12 is monomeric.

The Tyr21 peptide exhibits high specificity and affinity. We monitoredthe binding of unsulfated ₁₈SGDYDSM₂₄ (SEQ ID NO:5) and sulfated₁₈SGDsYDSM₂₄ (SEQ ID NO:4) to each CXCL12 variant by 2D NMR. Consistentwith the CXCL12₂:CXCR4₁₋₃₈ complex structure, the unsulfated peptideproduced chemical shift perturbations localized to residues in the β2strand, β3 strand and N-loop of each variant (FIG. 3A; FIG. 6).CXCL12_(WT) and CXCL12₁ further exhibited shifts in residues 58 and65-67 of the helix (FIG. 6). Sulfation of Tyr21 strengthened theaffinity of ₁₈SGDYDSM₂₄ (SEQ ID NO:5) for all three CXCL12 variants andincreased the magnitude of each perturbation without altering thepattern of responding residues (Table 1; FIG. 3B). The sTyr21sulfopeptide bound to CXCL12₂ with the highest affinity (K_(d)=211 μM)compared to all other peptide:chemokine combinations—four-fold strongerthan the unsulfated variant (K_(d)=831 μM).

Given that unsulfated CXCR4₁₋₃₈ (K_(d)=0.9 μM) binds CXCL12₂ with aGibbs free energy of −8.24 kcal mol⁻¹, our results suggest that the₁₈SGDYDSM₂₄ (SEQ ID NO:5) fragment alone provides about half of thebinding energy (ΔG=−4.2 kcal mol⁻¹) for the CXCR4:CXCL12₂ ‘site 1’interaction. The identification of such hot spots, or regions of bindinginterfaces that contribute substantial binding energy, are of particularinterest in the generation of protein-protein interface inhibitors. Themajor energetic contributions of the Tyr21 pocket justify our recentsuccess in developing small molecules that antagonize receptoractivation. Further, identification of the hydrophobic Tyr7 pocket onmonomeric CXCL12 demonstrates the utility of small receptor peptides inidentifying druggable chemokine hot spots.

Tyr7 and Tyr12 are dispensable for CXCR4 activation by CXCL12. TPSTenzymes catalyze the O-sulfation of Tyr21 much more efficiently than theTyr12 or Tyr7 residues in vitro. Further, sTyr21 recognizes a cleft onCXCL12 that may be conserved across most members of the chemokinesuperfamily. To test the hypothesis that Tyr21 sulfation is critical forreceptor activation, tyrosine to alanine mutations were introduced intoFLAG-tagged CXCR4 and expressed in CHO-K1 cells. CHO cells were chosenbecause they do not express endogenous CXCR4 and yield high levels ofsulfated protein. Receptor activation was assessed by monitoring thecalcium response as a function of increasing CXCL12_(WT) concentrations(FIG. 3C). The response of CXCR4(Y7A) was similar to wildtype CXCR4whereas the potency of CXCR4(Y12A) was reduced 3-fold. In contrast,CXCR4(Y21A) was significantly impaired both in terms of EC₅₀ andefficacy. The combined mutation of Y7A, Y12A, and Y21A did not furtherdiminish the potency relative to CXCR4(Y21A) but reduced the efficacy to˜20% of the wildtype receptor.

We hypothesize that protein misfolding is not responsible for thealtered efficacies for two reasons. First, all of the mutants aresurface-expressed at levels equivalent to the wildtype CXCR4 receptor(FIG. 9). Second, the CXCR4 N-terminus is disordered and is not believedto participate in folding the overall tertiary structure of thereceptor. This prompts the question of why there are efficacy changes atall. Our data suggests that the two-site model, in which site 1 isdiscretely responsible for chemokine binding and site 2 is specific foractivation, is oversimplified and that both of these regions areultimately required for full receptor activation. We conclude thatsulfotyrosine modifications serve both to enhance CXCL12 bindingaffinity, and therefore potency, as well as signaling efficacy. Takentogether, our results define the relative importance of individual CXCR4sulfotyrosine modifications (Tyr21>Tyr12>Tyr7) for CXCL12 binding andreceptor activation.

Binding at the Tyr21 site promotes dimerization. Chemokine dimerizationis highly sensitive to numerous factors including GAGs, divalent anions,and pH (32). The CXCR4 N-terminus also promotes CXCL12 dimerization,which drastically alters the cellular response. Interestingly, sTyr21sulfopeptide binding to CXCL12₂ is 20-fold stronger than to CXCL12_(WT)or CXCL12₁ (FIG. 3B). In addition, the sulfopeptide produces largeperturbations at the dimer interface of CXCL12_(WT) (FIG. 6) suggestingit may allosterically induce CXCL12_(WT) dimerization. We used intrinsictryptophan fluorescence polarization (FP) to determine the K_(d) forCXCL12_(WT) dimerization in the presence and absence of 3 mM sTyr21sulfopeptide. The sulfopeptide shifts the K_(d) for CXCL12_(WT)dimerization from 15.1±0.4 mM to 2.6±0.4 mM. Free sulfotyrosine(Tyr-SO₃H), which also binds the sTyr21 pocket, also promotes CXCL12dimerization (K_(d)=9.3±1.8 mM; FIG. 4A).

The free energy changes derived from NMR binding (ΔG_(bind)) and FPdimerization (ΔG_(dimer)) measurements at 298 K were used to construct athermodynamic cycle diagram (FIG. 4B). In pathway 1, CXCL12 binds thesTyr21 sulfopeptide (ΔG_(bind)=−3.8 kcal mol⁻¹) and then dimerizes(ΔG_(dimer)=−3.5 kcal mol⁻¹). When the sequence is reversed (pathway 2),CXCL12 dimerizes with ΔG_(dimer)=−2.5 kcal mol⁻¹ and then binds thesTyr21 sulfopeptide with ΔG_(bind)=−5.0 kcal mol⁻¹. Analysis of bothpathways yields similar coupling energies of −1.2 kcal mol⁻¹ and −1.0kcal mol⁻¹, respectively, that link CXCL12 dimerization to ligandbinding in the sTyr21 recognition pocket. Similar studies with both CCL5and CCL2 report binding of their respective N-terminal peptides, bothsulfated and unsulfated, to promote dimer dissociation. The apparentdisparity between these studies and our data is most easily explained bythe spatially distinct dimerization interfaces of CXC- and CC-typechemokines. CXCL12 dimerizes through the β1 strand and α-helix whereasCC-type chemokines self-associates using the N-terminus, N-loop and β3strand. Nonetheless, both chemokines bind sulfopeptides in the cleftformed by the N-loop and β3 strand. The close proximity of thesulfopeptide-binding site to the CC-type dimer interface is moreconsistent with dissociation through direct binding competition ratherthan an allosteric mechanism.

Many intracellular signal transduction complexes involve recognition ofa recurring sequence motif by a protein interaction domain. Binding tocertain short linear motifs (or SLiMs) depends on post-translationalmodifications (PTMs) like phosphorylation, acetylation or methylation,and their corresponding recognition sites are often viewed as promisingtargets for drug discovery. Likewise, tyrosine sulfation enhancesprotein-protein interactions in the extracellular space andsulfotyrosine recognition likely defines a new class of druggableextracellular targets. However, selective binding may require thecombination of multiple SLiMs as observed for the WASP interactingprotein (WIP) which uses three distinct recognition epitopes, includingthe conserved polyproline motif, to bind the EVH1 domain of N-WASP.

Many chemokine receptors contain multiple N-terminal tyrosines, whichare predicted to be sulfated to different levels based on thesuitability of the flanking sequences for TPST recognition. We treatedeach tyrosine in the CXCR4 N-terminus as the center of a SLiM and foundthat the most efficient site of enzymatic sulfation (Tyr21) is also themost important motif for binding to both CXCL12 monomers and dimers.Surprisingly, neither the Tyr7 or Tyr12 motif bound to a unique site onthe CXCL12 dimer, and sulfation of the Tyr7 motif eliminated thesite-specific binding to the CXCL12 monomer observed with the unsulfatedTyr7 motif. To our knowledge, this is the first demonstration thatsulfation of chemokine receptors does not universally enhance complexaffinity.

In contrast, our data suggests that distinct patterns of tyrosinesulfation could encode selectivity either by enhancing or reducingaffinity for unique recognition sites. Taken together, this impliesanother layer of regulation for chemokine signaling. The structuralbasis for this effect awaits further study, but it appears thatmonosulfated (Tyr7/Tyr12/sTyr21) CXCR4 would exhibit a preference forthe monomeric CXCL12 ligand while sulfation of Tyr7 would bias CXCR4toward interactions with a CXCL12 dimer. Regardless, we conclude thatthe most functionally relevant mode of CXCL12-CXCR4 interaction involvesspecific recognition of a sulfotyrosine at position 21, and additionalinteractions with the receptor N-terminus that are independent oftyrosine sulfation.

Our results for CXCL12 binding to the sTyr21 motif in CXCR4 also suggestthat chemokine oligomerization may be subject to allosteric control.Binding of the sTyr21 sulfopeptide, which is too short to contact bothsubunits of a CXCL12 dimer, significantly enhances CXCL12 dimerizationby an indirect mechanism. Because dimerization converts CXCL12 into apartial agonist that potently inhibits chemotaxis and tumor metastasis,variations in the sulfation pattern of CXCR4 could in principle havefunctional consequences in vivo. Moreover, ligands that occupy thesTyr21 recognition site of CXCL12 may act as competitive inhibitors ofreceptor binding and allosteric modulators of chemokine function.

Construction of CXCL12₁ Plasmid.

The CXCL12₁ variant was produced via mutagenesis of the CXCL12_(WT)construct using complementary primers and the QuikChange Site-DirectedMutagenesis Kit (Stratagene) as per the manufacturer's instructions. Theexpression vector insert was confirmed by DNA sequencing.

Protein Expression and Purification.

CXCL12_(WT), CXCL12₁ and CXCL12₂ were expressed and purified asdescribed previously. CXCR4₁₋₃₈, comprising the first 38 amino acids ofCXCR4 preceded by a residual GlyMet dipeptide tag, was expressed andpurified as previously described.

Size-Exclusion Chromatography Multi-Angle Light Scattering (SEC-MALS).

SEC was performed at a flow rate of 0.4 ml min⁻¹ on a Superdex 75 10/300GL analytical column (GE Healthcare) and monitored by an 18-angle MALSdetector (Dawn Heleos II). CXCL12₁ (40 mg ml⁻¹; 5 mM) and CXCL12₂ (40 mgml⁻¹; 2.5 mM) were solubilized in 25 mM MES (pH 6.8), 650 mM NaCl, and0.02% (w/v) NaN₃. Samples were then applied to the column in a mobilephase of identical composition. Peak dispersion and average molar masswere calculated using Astra software.

NMR Spectroscopy.

All NMR spectra were acquired on a Bruker DRX 600 MHz spectrometerequipped with a ¹H, ¹⁵N, ¹³C TXI cryoprobe at 298 K. Experiments wereperformed with either 50 μM [U-¹⁵N]-CXCL12_(WT), -CXCL12₁, or -CXCL12₂proteins in a solution containing 25 mM deuterated MES (pH 6.8), 10%(v/v) D₂O, and 0.02% (w/v) NaN₃. Full-length CXCR4₁₋₃₈ peptidetitrations required 20 μM [U-¹⁵N]-CXCL12₁ and -CXCL12₂ protein samples.Sulfated CXCR4 peptides were reconstituted at 100 mM in 25 mM deuteratedMES, 10% (v/v) D₂O, and 0.02% (w/v) NaN₃ buffer. Unsulfated CXCR4peptides were similarly reconstituted to 15 mM. Two separate ₄ISIYTSD₁₀(SEQ ID NO:8) peptide samples were reconstituted at 10 mM and 15 mMpeptide in 25 mM deuterated MES, 10% (v/v) D₂O, 0.02% (w/v) NaN₃ buffer,from lyophilized powder. The 10 mM batch of ₄ISIYTSD₁₀ (SEQ ID NO:8)peptide was titrated into CXCL12_(WT) and CXCL12₂ and the 15 mM batchwas titrated into CXCL12₁. CXCL12_(WT) and CXCL12₂ chemical shiftassignments (H and ¹⁵N) were acquired from previously published sources(BMRB ID 16145 and 15633, respectively).

Peptides were titrated into CXCL12 samples and monitored by ¹H-¹⁵NHeteronuclear Single Quantum Coherence (HSQC) experiments. Total peptideadditions differed for individual peptide titrations but ranged from0-160 equivalencies. Spectra were processed using in-house scripts andchemical shift tracking was performed using CARA software. Combined¹H/¹⁵N chemical shift perturbations were calculated as ((5Δδ_(H))²+(Δδ_(NH))²)^(0.5), where ⁶H and ⁶NH are the amide proton andnitrogen chemical shifts, respectively. Equilibrium dissociationconstants (K_(d)) were determined by non-linear fitting of the combined¹H/¹⁵NH chemical shift perturbations as a function of peptideconcentration to a single-site quadratic equation. For a giveninteraction, residues with the largest chemical shift perturbations werefitted individually. The resulting K_(d) values and their respectivefitting errors were then averaged to produce the reported affinity andstandard deviation for that interaction.

Peptide HPLC Purification.

HPLC was performed on a Waters 600E multisolvent delivery system with aWaters U6K injector, Waters 490E programmable multi-wavelength detectoroperating at 230 nm, Waters busSAT/IN module and Waters Empower 2software. Separation was achieved on a Sunfire™ SemiPrepC₁₈ OBS™ column(5 μm, 150×19 mm ID) at a flow rate maintained at 7.0 mL min⁻¹, or on aSunfire™ SemiPrepC₁₈ OBS™ column (5 μm, 250×10 mm ID) at a flow ratemaintained at 4.0 mL min⁻¹. Method A: Separation of non-sulfatedpeptides and neopentyl-protected sulfopeptides involved a mobile phaseof 0.1% TFA in water (Solvent A) and 0.1% TFA in acetonitrile (SolventB) using a linear gradient from 0% to 100% solvent B. Method B:Separation of unprotected sulfopeptides involved a mobile phase of 0.1 MNH₄OAc (Solvent C) and 100% acetonitrile (Solvent D) using the statedlinear gradient.

Peptide Synthesis—Loading of Amino Acid onto Rink Amide Resin.

Solid-phase synthesis was carried out in polypropylene syringes equippedwith Teflon filters, purchased from Torviq. Rink Amide resin (resincapacity 0.74 mmol g⁻¹) (200 μmol) was initially washed with DMF (5×5mL), DCM (5×5 mL) and DMF (5×5 mL), then allowed to swell in DMF (5 mL)for 30 min. The resin was drained and treated with a solution ofpiperidine/DMF (2×5 mL, 1:9 v/v) and gently agitated for 5 min. Theresin was then washed sequentially with DMF (5×5 mL), DCM (5×5 mL) andDMF (5×5 mL). The efficiency of the deprotection was determined bymeasurement of the fulvene-piperidine adduct at λ=301 nm (see below). Asolution of protected amino acid (800 μmol), PyBOP (416 mg, 800 μmol)and NMM (16 μL, 800 μmol) in DMF (8 mL) was added to the resin and theresulting suspension was gently agitated for 2 h. At this time the resinwas drained and washed with DMF (5×5 mL), DCM (5×5 mL) and DMF (5×5 mL).A mixture of acetic anhydride/piperidine (5 mL, 1:9 v/v) was then addedto the resin and agitated for 10 min. The resin was subsequently drainedand washed with DMF (5×5 mL), DCM (5×5 mL) and DMF (5×5 mL).

Peptide Synthesis—Estimation of Resin Loading.

The N-terminal Fmoc protecting group was removed according to theprocedure described above and the combined drained Fmoc deprotectionsolutions were diluted with a solution of piperidine/DMF (1:9 v/v) sothat the maximum concentration of the fulvene-piperidine adduct was inthe range of 2.5-7.5×10⁻⁵ M. A sample of this solution (2×3 mL) wastransferred to two matched 1 cm quartz glass cuvettes and the UV-Visabsorbance at λ=301 nm was measured using the solution of piperidine/DMF(1:9 v/v) as a reference. An average of the two absorbance values wereused to calculate the resin loading using ε=7800 M⁻¹ cm⁻¹

Peptide Synthesis—Iterative Fmoc-Strategy Peptide Assembly (100 μmol).

N-terminal Fmoc deprotection: A solution of piperidine/DMF (2×5 mL, 1:9v/v) was added to the resin and agitated for 5 min. the resin wassubsequently drained and washed with DMF (5×3 mL), DCM (5×3 mL) and DMF(5×3 mL). The resulting resin-bound amine was used immediately in thenext peptide coupling step. The efficiency of the previous amino acidcouplings were determined by measurement of the resultingfulvene-piperidine adduct at λ=301 nm, as described above. Amino acidcoupling: a solution of Fmoc-protected amino acid (100 μmol), PyBOP (208mg, 400 μmol) and NMM (22 μL, 200 μmol) in DMF (1 mL) was added to theresin and the resulting suspension was gently agitated for 1 h. Theresin was then drained and washed sequentially with DMF (5×3 mL), DCM(5×3 mL), and DMF (5×3 mL). Fmoc-Tyr(SO₃nP)—OH (prepared as describedpreviously (35, 36)), was coupled using 1.5 equiv. of the modified aminoacid, PyBOP (1.5 equiv.) and NMM (3 equiv.) in DMF (1 mL), and thesuspension was gently agitated for a minimum of 4 h. Capping: a mixtureof acetic anhydride/pyridine (2×5 mL, 1:9 v/v) was added to the resinand agitated at room temperature for 5 min. At this time the resin wasdrained and washed with DMF (5×3 mL), DCM (5×3 mL) and DMF (5×3 mL).Final N-terminal Fmoc deprotection and acetylation: the fully assembledresin bound peptide was treated with piperidine/DMF (2×5 mL, 1:9 v/v)and agitated for 5 min, The loading was determined by measuring theabsorbance of the fulvene-piperidine adduct at λ=301 nm. The N-terminuswas subsequently acetylated by treatment with a mixture of aceticanhydride/pyridine (5 mL, 1:9 v/v) for 5 min and the resin subsequentlywashed with DMF (10×3 mL) then DCM (10×3 mL).

Peptide Synthesis—General Procedure for Cleavage of Peptides andProtected Sulfopeptides from Resin.

A mixture of TFA/triisopropylsilane/water (3 mL, 90:5:5 v/v/v) [orTFA/triisopropylsilane/water/thioanisole (3 mL, 85:5:5:5 v/v/v/v) forpeptides containing methionine residues] was added to the resin and theresulting suspension gently agitated for 2 h. The resin was then drainedand washed with TFA (3×3 mL) and the combined cleavage/washing solutionswere concentrated under reduced pressure. The resulting residue wasdissolved in water (2 mL) with dropwise addition of MeCN to facilitatedissolution. Purification by preparative reverse-phase HPLC (method A)and concentration of the appropriate fractions provided the desiredpeptides and neopentyl-protected sulfopeptides.

Peptide Synthesis—General Procedures for Deprotection of NeopentylProtected Sulfopeptides.

Method A: Neopentyl-protected sulfopeptides (100 μmol) were dissolved in1 M NH₄OAc solution (˜2 mL/10 mg peptide) and the resulting mixture wasincubated at 310 K for 24 h. At this time, the reaction mixture waspurified by preparative reverse-phase HPLC (Method B). Lyophilization ofthe appropriate fractions provided the desired free sulfopeptides.Lyophilization was performed three times until a constant weight wasachieved suggesting complete removal of residual NH₄OAc. Method B: To asolution of neopentyl protected sulfopeptide (100 μmol) in a mixture ofDMF/DMSO (˜1 mL/10 mg peptide, 3:1 v/v) was added sodium azide (1000μmol) and the resulting mixture was stirred at 338 K for 12 h under anatmosphere of nitrogen. The reaction mixture was subsequently purifiedby preparative reverse-phase HPLC (Method B). Lyophilization of theappropriate fractions provided the desired sulfopeptides. Thesulfopeptides were resuspended in MilliQ water and lyophilized threetimes to remove residual NH₄OAc.

Fluorescence Polarization Assay.

Fluorescence polarization (FP) assays were performed on a PTIspectrofluorometer equipped with automated polarizers, using a time basepolarization method provided by the program Felix32. LyophilizedCXCL12_(WT) was reconstituted in H₂O and diluted to appropriateconcentrations in a 25 mM MES (pH 6.8) buffer, filtered and degassed.₁₈SGDsYDSM₂₄ (SEQ ID NO:4) peptide and sulfotyrosine (Tyr-SO₃H) stockswere prepared separately at 25 mM and 500 mM, respectively, in a 25 mMMES (pH 6.8) buffer, filtered and degassed. Experiments were performedat 298 K and intrinsic tryptophan fluorescence was observed withemission and excitation wavelengths of 325 nm and 295 nm, respectively.FP was monitored as a function of increasing CXCL12_(WT) concentration(10, 25, 50, 75, 100, 250, 500, 750, 1000 and 1500 μM) alone or in thepresence of 3 mM ₁₈SGDsYDSM₂₄ (SEQ ID NO:4) or 50 mM sulfotyrosine. TheCXCL12_(WT) dimerization equilibrium dissociation constant (K_(d)) wasdetermined by non-linear fitting to a three-parameter function aspreviously described. Experiments were performed in duplicate and thereported dimerization K_(d) values reflect the average of bothexperiments.

Cell Culture.

Chinese hamster ovary K1 (CHO-K1) cells, stably transfected with theGα15 gene in pcDNA3.1+, were cultured in a 1:1 mixture of Dulbecco'smodified Eagle medium (DMEM) with Glutamax (Gibco):F12 nutrient mixture(Gibco) supplemented with 10% (w/v) fetal bovine serum (FBS) (Gibco).Stable expression of the Gα15 transgene was maintained by furthersupplementing the growth medium with 700 μg ml⁻¹ geneticin (Gibco).

Transfections.

For transient transfections with the Flag-CXCR4 WT and N-terminallymutated constructs, the CHO-K1 Gα15 stable cells were lifted using 0.25%(v/v) trypsin-EDTA (Gibco), and 2×10⁶ cells were re-plated onto 10 cmculture dishes. To increase transfection efficiency, 0.25% (v/v) DMSOwas added to the media when the cells were plated, and no geneticin waspresent in the plating medium. The cells were transfected 24 hours laterusing the Mirus TransIT-CHO Transfection Kit according to themanufacturers protocol, with the following exceptions: the amount of CHOtransfection reagent was scaled up to 4 μl μg⁻¹ of DNA in thetransfection mixtures, and the amount of CHO mojo reagent was scaled upto 1 μl μg⁻¹ of DNA in the mixtures. Immediately before transfection,the medium was replaced with a 1:1 mixture of DMEM with Glutamax:F12nutrient mixture supplemented with 10% (w/v) FBS (without DMSO orgeneticin).

Calcium Flux.

24 hours after plating, the medium was removed and the cells were washedwith 5 ml PBS. The adherent cells were then incubated for 10 minutes in3 ml of Cellstripper non-enzymatic cell dissociation solution (Cellgro).Cells were then suspended by pipetting, washed twice in Calcium FluxBuffer (Hanks Balanced Salt Solution supplemented with 20 mM HEPES)supplemented with 0.1% (w/v) bovine serum antigen (BSA) and 4 mMprobenecid (Invitrogen). Each washing was carried out by centrifugingthe cells at 350×g and then re-suspending in Calcium Flux Buffer. Afterwashing, the cell concentrations were normalized between samples and thecells were plated at 2.5×10⁵ cells/well in poly-D-lysine coated 96 wellplates (Becton Dickinson Labware). FLIPR 4 Calcium Flux assay kit dye(Molecular Devices) was then added to each well, such that the ratio ofdye to cell suspension was 1:1. The plates were then centrifuged for 3minutes at 250×g to ensure the cells settled onto the surface of theplates. The plated cells were incubated at 310 K for 90 min.Fluorescence was measured at 310 K using a FlexStation2 MicroplateReader with excitation and emission wavelengths at 485 nm and 515 nm,respectively. After an 18 s baseline measurement, the indicatedconcentrations of CXCL12 were added and the resulting calcium responsewas measured for an additional 50 s. Fluorescence as a function ofCXCL12 concentration was fitted to a four-parameter equation. Data arerepresentative of two experiments each performed with three replicates.CXCR4 variants with EC₅₀ or maximum calcium response values more thanthree standard deviations from the mean CXCR4_(WT) quantities weredeemed significant.

Flow Cytometry.

For testing receptor surface expression, 3×10⁵ cells were set-asideduring the calcium flux assay preparations. The cells were then washedtwice with PBS containing 0.5% BSA (w/v) (FACS buffer). Staining wascarried out in a 50× dilution of either anti-DDDDK or mouse IgG1 isotypecontrol conjugated to SureLight APC (Columbia Biosciences) for 45minutes on ice. Cells were then washed 3× in FACS buffer beforeanalysis, which was carried out using a Guava bench top mini-flowcytometer.

Example 2: Complex Structure of Chemokine-GPCR N-Terminus: Insight intoCXCL12 Biased Agonism

Chemokines are ubiquitous in the directed migration of cells fordevelopment, immune surveillance and disease. Approximately twentyreceptors and more than twice that number of chemokine ligands create acomplex system to regulate the appropriate physiologic response. Thenetwork is further complicated by receptor and ligand promiscuity, andmore recently the discovery of biased agonism. The receptor CXCR4recognizes both monomeric and dimeric forms of the chemokine CXCL12, butthe structural mechanism for biased agonism remains unclear. We solvedthe structure of a 1:1 complex between CXCL12 and the CXCR4 N-terminusand observed a monomer-specific interaction that is required forreceptor activation. Measurements of CXCL12-dependent arrestinrecruitment, receptor modification, trafficking and degradation, cellmigration and apoptotic cell death revealed distinct CXCR4 activationprofiles that correlate with binding of the monomeric balanced agonistor dimeric arrestin-biased agonist.

The last decade witnessed major revisions to the classic model of Gprotein-coupled receptor (GPCR) signaling. Instead of a single type ofagonist-driven intracellular response, different ligands can stabilizedistinct active states in the same receptor to shift the balance offunctional outputs. The predominant modes of GPCR signaling originatewith GTPase activation and β-arrestin recruitment, and agonists canselectively activate one (‘biased agonists’) or both (‘balancedagonists’) pathways. In the chemokine family where receptor and ligandpromiscuity is common, biased agonist GPCR signaling adds another layerof complexity to the in vivo orchestration of cell migration. Originallydiscovered as leukocyte chemoattractants that promote inflammation anddirect the trafficking of immune cells, the biological responsibilitiesof chemokines include organogenesis, neurogenesis, would healing, andcardioprotection. In a family of more than 50 chemokines and 19receptors, the chemokine CXCL12 (Stromal cell-derived factor-1; SDF-1)and its receptor CXCR4 have been the focus of intense study for over twodecades. CXCL12 and CXCR4 are essential in developmental andhousekeeping roles but also participate in numerous pathologiesincluding HIV infection and more than 23 different types of cancer.Despite numerous efforts to inhibit CXCL12/CXCR4 signaling, only oneantagonist, AMD3100, is currently approved for clinical use. A betterpharmacological understanding based on GPCR-ligand structure-functionrelationships is needed as a foundation for next-generation discoveryresearch on this important therapeutic target.

Chemokine signaling is initiated by formation of an extensiveprotein-protein interface segregated into two distinct regions. First,the receptor N-terminus wraps around the folded chemokine domain andcontributes most of the binding energy (site 1), and solublechemokine-receptor complexes corresponding to the site 1 interactionhave been analyzed by NMR (REFS). Subsequent docking of the flexiblechemokine N-terminus into a pocket within the transmembrane domainactivates receptor signaling (site 2). X-ray crystal structures ofinhibitor-bound CXCR4 reveal the large orthosteric pocket but lackelectron density for the receptor N-terminus. Thus, no structure of anintact, active chemokine-receptor complex has been solved.

Like most chemokines, CXCL12 forms dimers at high concentrationsVeldkamp 2006, and www.ncbi.nCXCL121.nih.gov/pubmed/17075134), whencrystallized, or when bound to extracellular matrix glycosaminoglycans,but is presumed to bind CXCR4 as a monomer at chemotactic concentrations(˜10 nM). However, we found that peptides derived from the CXCR4N-terminus bind the CXCL12 monomer and also form a dimeric 2.2 complexat higher concentrations. We subsequently showed thatpreferentially-monomeric and constitutively-dimeric CXCL12 variantsactivate distinct intracellular signaling and migration profiles, aunique example of biased agonism arising from a change in oligomericstate of the ligand. Some of the contacts observed in the dimericCXCL12-CXCR4₁₋₃₈ NMR structure would be absent in a monomeric complex,suggesting that distinct monomer- and dimer-specific contacts at theCXCL12-CXCR4 site 1 interface contribute to biased signaling.

To examine the structural basis for biased CXCR4 agonism, we solved thestructure of a monomeric CXCL12 variant (CXCL12₁ bound to the N-terminaldomain of CXCR4 (CXCR4₁₋₃₈). Apolar residues near the CXCR4 N-terminusdock into a cleft on the CXCL12 monomer that is inaccessible in thedimer, and this monomer-specific interaction is essential for fullreceptor activation. Functional comparisons of receptor activation byconstitutively monomeric and dimeric ligands show that only the CXCL12monomer induces CXCR4 phosphorylation at Ser 324 and Ser 325,ubiquitination, and degradation as well as the induction of apoptoticcell death. These results suggest that the two-site model for chemokinereceptor activation must evolve to account for ligand-biased agonismarising from alternative site 1 interfaces.

CXCL12₁ binds and activates the CXCR4 chemokine receptor. For nearly twodecades, the CXCL12 quaternary structure relevant to receptor activationhas been debated. We and others have shown that CXCL12 exists in asolution-dependent dimer equilibrium that effects binding toglycosaminoglycans, receptor recognition and ultimately modulatesintracellular signaling. Subsequent studies attempting to deconvolutethe effects of dimerization have compared constitutively dimeric CXCL12(CXCL12₂) to either wildtype (CXCL12_(WT)) or a preferential-monomer. Werecently engineered a CXCL12 variant that remains strictly monomeric atmillimolar concentrations (CXCL12₁).

Here, we tested whether the affinity of CXCL12₁ for full-length CXCR4differed from CXCL12_(WT). Radioligand displacement using CXCR4 cellmembrane preparations yielded similar affinities for CXCL12_(WT) andCXCL12₁ of 1.44±1.5 nM and 0.97±1.5 nM, respectively (FIG. 10A). Weestablished the functional activity of CXCL12₁ as a CXCR4 agonist bymeasuring its ability to mobilize intracellular calcium, a sensitiveindicator of chemokine receptor-mediated G protein activation. CXCL12₁produced a robust calcium flux response with a potency equivalent toCXCL12_(WT) (FIG. 10B).

CXCL12₁ has enhanced migratory capacity. CXCL12, like all chemokines,promotes a bell-shaped migratory response over a narrow concentrationrange. Although the mechanism is unclear, we suggested that the CXCL12monomer-dimer equilibrium might be partially responsible for the narrowdose response. Whereas CXCL12₂ does not promote chemotaxis at anyconcentration, a preferentially monomeric CXCL12 variant produces abell-shaped response over an extended concentration range. BecauseCXCL12₁ is incapable of dimerization, we tested if it generated asigmoidal or biphasic migratory response. The chemotaxis of THP-1monocytes, NACXCL1216 pre-B cells, and MiaPaCa2 pancreatic cancer cellswas tested using Boyden or Transwell migration chambers (FIG. 10C-E).CXCL12₁ extended the effective dose at least 10-fold in all cell typesbut, nonetheless, still signaled migratory arrest.

To assess how CXCR4 activation was interpreted in the presence of othermigratory signals, U-937 leukemia cells confined to an agarose dropletwere incubated in serum-containing media with increasing CXCL12concentrations. CXCL12₁ significantly enhanced migration at all testedconcentrations (FIG. 10F). Qualitatively, CXCL12₁ exhibits a bell-shapeddose response with a peak enhancement at 25 nM. In stark contrast,CXCL12_(WT) prompted dose-dependent migratory arrest at nearly allconcentrations with maximal inhibition of 40.1±18% (FIG. 10F).

CXCR4-mediated chemotaxis is dependent upon β-arrestin recruitment andfilamentous actin cytoskeleton rearrangement. Consistent with itsnon-migratory phenotype, CXCL12₂ produces little arrestin recruitment orF-actin formation. In contrast, CXCL12_(WT) and CXCL12₁ recruitβ-arrestin-2 to CXCR4 with similar potency and efficacy (FIG. 10G).Interestingly, as concentrations were increased to 10 and 100 μMCXCL12_(WT) induced a bimodal recruitment of β-arrestin-2 that parallelsthe chemotactic dose response. As CXCL12₁ is incapable of dimerization,our data suggests that ligand-receptor stoichiometry, independent of thechemokine quaternary structure, may govern CXCR4 signaling.

The structure of CXCL12₁ in complex with CXCR4₁₋₃₈. The dimeric CXCR4crystal structures reinforce the hypothesis that ligand-receptoroccupancy may modulate CXCR4 signaling bias. In the two-step, two-sitemodel for chemokine receptor activation, CXCL12 first binds to theN-terminus of CXCR4. Our recent 2D NMR studies suggest that theCXCL12₁:CXCR4₁₋₃₈ interface is partially distinct from the 2:2 site 1interaction. We used HSQC titrations to confirm the 1:1 interaction.Increasing CXCL12_(WT) concentrations caused CXCR4₁₋₃₈ resonances toshift in curved trajectories suggesting the simultaneous presence ofboth a 1:1 and 2:2 complexes. Titrating CXCL12 variants into[U-¹⁵N]-CXCR4₁₋₃₈ produced chemical shift perturbations consistent withunique chemical environments for CXCR4 residues 1-13 (FIG. 12B).

In contrast to previously published spectra of CXCL12_(WT), all peaks inthe N-terminus were visible throughout the titration and traversedlinear paths. Concentration of the linear trajectories recreates thecomplicated chemical shifts induced by CXCL12_(WT) and underscores thephysiologic validity of both the CXCL12₁ and CXCL12₂ variants (FIG.16B). To assess the rigidity of CXCR4₁₋₃₈ upon chemokine binding wemeasured ¹H-¹⁵N heteronuclear NOE values, which reflect the backboneflexibility for each residue on picosecond to nanosecond timescales.When bound to the CXCL12₂ dimer, CXCR4₁₋₃₈ residues 5-10 remainrelatively dynamic and imply a weak, transient interaction. CXCL12₁stabilizes the high frequency motions of CXCR4₁₋₃₈ residues 5-10 (FIG.16A).

To understand how the CXCR4 N-terminus recognizes CXCL12₁, we determinedthe complex structure by NMR. A suite of triple-resonance experimentswere acquired on samples of [U-¹³C,¹⁵N]-CXCL12₁ saturated with CXCR4₁₋₃₈and [U-¹³C,¹⁵N]-CXCR4₁₋₃₈ saturated with CXCL12₁ to assign the carbon,nitrogen, and hydrogen resonances. Next, a series of 3D nuclearOverhauser effect (NOE) spectroscopy (NOESY) experiments were collectedto identify the position of neighboring hydrogen atoms within a 5Adistance. As expected, CXCL12₁ adopts the canonical chemokine foldcomprising a flexible N-terminus, followed by the N-loop, athree-stranded antiparallel β-sheet, and a C-terminal α-helix (FIG. 11).CXCL12₁ was designed to mimic the conformation of PDB 1SDF, a CXCL12solution NMR structure determined in sodium acetate buffer at pH 4.9. Inthis structure the α-helix maintains a 55 relative to the β-sheet, whichwould be sterically incompatible with dimerization. As illustrated inFIG. 14, the CXCL12₁ α-helix is ˜65° relative to the β-sheet and alignsto PDB 1SDF with a backbone RMSD of 1.6 Å.

Determination of the CXCR4₁₋₃₈ contact surface requiredF1-¹³C-filtered/F3-¹³C-edited NOESY-HSQC experiments to unambiguouslyidentify intermolecular constraints. Intermolecular NOEs, indicative ofa stable interaction on the millisecond timescale, were observed fromresidues 4-27 along the peptide (FIG. 11). Overall, CXCR4₁₋₃₈ adopts arandom coil architecture with a short β-strand from Tyr7-Ser9. Theposition of CXCR₁₋₃₈ overlaps well with the previously publishedchemical shift perturbations (FIGS. 11A-C). The ProCys motif (CXCR4Pro27 and Cys28) is nearly conserved across all chemokine receptors anddelineates the flexible N-terminus from the transmembrane portion. Herewe observed intermolecular NOEs from both the CXCL12 N-loop and helix toCXCR4 Lys25, Glu26, and Ala28. This constrains CXCR4 Pro27 between thehelix and N-loop, marking the point where our NMR data intersects withthe recent CXCR4 crystal structure. The Pro27 Ca is translated anaverage 8.6 Å toward the N-loop compared to the 2:2 complex.Subsequently, the pocket formed by the N-loop and β3 strand, known asthe chemokine cleft, is slightly distorted. CXCL12₁ residues Glu15-His17shift toward the β3 strand to accommodate the peptide. CXCL12₁ Asn46puckers away from the pocket allowing Arg47, and to a lesser extentAns45, to interact with the CXCR4 Tyr21 hydroxyl. The C^(ζ) and C^(α)atoms of Tyr21 are translated an average of 7.0 Å and 5.3 Å,respectively, compared to the 2:2 complex (FIG. 11D). In this positionthe Tyr21 hydrophobic contacts appear to be primarily satisfied by Val49and the methylene of Glu15. Upon sulfation, it's reasonable to predictthat Lys47, Asn45 or His17 maintain sTyr21 electrostatic interactionswith negligible pocket rearrangement.

The 1:1 and 2:2 complex interfaces sharply diverge at residuesN-terminal of CXCR4 Tyr12. In the 2:2 complex, CXCR4 Tyr12 makeselectrostatic contacts with Lys27 of one CXCL12₂ protomer and His25 ofthe other. Upon sulfation, sTyr12 preferentially forms salt bridges withLys27 from a single protomer. Tyr7 also makes contacts with the otherCXCL12₂ subunit by burying into a pocket formed by Val23 and Arg20. Inthe 1:1 complex Tyr12 buries into a deep cleft formed by Pro10, Lys27,Leu29, and Val 39 that possesses no obvious electrostatic or chargeinteractions for a sulfated tyrosine. Asp10 and Asn11 of CXCR4₁₋₃₈ thenturn to place Tyr7 in close proximity to Tyr12. The Tyr7 hydroxyl ispositioned toward His25 and Lys27, but productive contacts are notclear. CXCR4₁₋₃₈ residues Tyr7-Ser9 hydrogen bond with CXCL12₁Ile28-Asn30 to form a four-stranded antiparallel β-sheet (FIG. 11E andFIG. 15).

The position of Tyr7 enables Ile6 to bury into a hydrophobic cleftsurrounded by Leu26, Tyr61, and Ala65 (FIG. 11F). Ile4 packs further upthe helix against CXCR4 Ile6 and CXCL12₁ Leu26, Trp57, and Tyr61. Theinteraction of hydrophobic CXCR4 residues with the CXCL12 helix isconsistent with previous NMR titration studies of CXCR4₁₋₃₈ andcross-saturation NMR experiments with full-length CXCR4. Sulfation ofTyr7 reduces the binding affinity of a CXCR4 Ile4-Asp10 heptapeptidetwo-fold. The reduced affinity may result from a sulfated Tyr7 strainingto interact with Lys27 and displacing the isoleucines from theirhydrophobic cleft.

The CXCR4 N-terminus is critical for chemokine recognition andactivation. The structure of CXCR4₁₋₃₈ in complex with CXCL12₁ containsa unique interface for residues 1-12 compared to the CXCL12₂:CXCR4₁₋₃₈complex (FIG. 11G). To assess the contributions of CXCR4 Ile4 and Ile6to receptor binding and activation, we designed a series of CXCR4mutants. Ile4 and Ile6 were simultaneously mutated to either alanine orglutamine residues in FLAG-tagged CXCR4. Whereas alanine substitutionhad no effect on binding affinity, the affinity of CXCL12_(WT) andCXCL12₁ for glutamine mutants was reduced 30- and 90-fold, respectively(FIG. 17). Receptor activation was then monitored by the calciumresponse in CHO-KI cells. Alanine substitutions had no effect on theCXCR4 signaling consistent with an apolar binding interaction (FIG. 17).In contrast, mutation to charged side chains reduce the EC₅₀ nearly10-fold (FIG. 17, Table 2).

TABLE 2 NMR refinement statistics of CXCL12₁:CXCR4₁₋₃₈ 20 modelensemble. Experimental constraints Distance constraints Intermolecular72 Long 414 Medium [1 < (i − j) ≤ 5] 241 Sequential [(i − j) = 1] 441Intraresidue [i = j] 365 Total 1533 Dihedral angle 109 constraints (φand ψ) Average atomic R.M.S.D. to the mean structure (Å) CXCL12₁residues 9-66 and CXCR4₁₋₃₈ residues 4-27 Backbone 3.24 ± 0.73 (C^(α),C′, N) Heavy atoms 4.07 ± 0.73 Deviations from idealized covalentgeometry Bond lengths RMSD (Å) 0.018 Torsion angle violations RMSD (°)1.4 WHATCHECK quality indicators Z-score −3.12 ± 0.27  RMS Z-score Bondlengths 0.78 ± 0.03 Bond angles 0.77 ± 0.02 Bumps 0 ± 0 Lennard-Jonesenergy ^(a) −2262 ± 99   (kJ mol⁻¹) Constraint violations ^(b, c) NOEdistance Number >0.5 Å 0 ± 0 NOE distance RMSD (Å) 0.0196 ± 0.0013Torsion angle violations Number >5° 0 ± 0 Torsion angle violations RMSD(°) 0.6474 ± 0.1020 Ramachandran statistics (% of all residues) Mostfavored 73.1 ± 2.9  Additionally allowed 22.3 ± 3.6  Generously allowed2.96 ± 2.0  Disallowed 1.57 ± 1.2  ^(b) Final X-PLOR force constantswere 250 (bonds), 250 (angles), 300 (impropers), 100 (chirality), 100(omega), 50 (NOE constraints), and 200 (torsion angle constraints). ^(c)Nonbonded energy was calculated in XPLOR-NIH. ^(d)The largest NOEviolation in the ensemble of structures was 0.274 Å.

Monomeric CXCL12 mediates CXCR4 degradation, phosphorylation andubiquitination. We recently demonstrated that CXCR4 signaling exhibitsagonist bias for wildtype and dimeric CXCL12. β-arrestin-2 signaling wasdiminished and β-arrestin-2 is necessary for many G protein independentsignaling. In addition to desensitization, arrestins mediate secondarysignaling and ubiquitination. To investigate the effect of CXCL12oligomeric state on CXCR4 trafficking, we initially examined CXCR4lysosomal targeting and degradation. HeLa cells were treated for 3 hwith 80 ng/ml CXCL12, CXCL12₁, and CXCL12₂ and receptor levels weredetected by immunoblot analysis, as previously described. Both CXCL12and CXCL12₁ promoted approximately 60% CXCR4 degradation compared tovehicle treated cells. In contrast, degradation promoted by CXCL12₂ wassignificantly reduced (˜15% vs 60%; FIG. 12A). Internalization is aprerequisite for degradation; therefore, we next examined the ability ofthese ligands to promote CXCR4 internalization. HeLa cells were treatedwith CXCL12, CXCL12₁, and CXCL12₂ for 20 min followed by FACS to measureCXCR4 surface expression. CXCL12₁ promoted CXCR4 internalization to thesame levels as that promoted by CXCL12 (FIG. 12B). However,internalization promoted by CXCL12₂ was significantly consistent withinefficient degradation (FIG. 12B).

Ubiquitination of CXCR4 by the E3 ubiquitin ligase AIP4 oncarboxy-terminal lysine residues is essential for its targeting anddegradation in lysosomes. We next examined the effect of CXCL12,CXCL12₁, and CXCL12₂ on CXCR4 ubiquitination. HEK293 cells stablyexpressing HA-CXCR4 and transfected with FLAG-ubiquitin were treatedwith vehicle and each CXCL12 variant for 30 min. HA-CXCR4 wasimmunoprecipitated and ubiquitinated receptor was detected byimmunoblotting for the FLAG epitope to detect incorporated ubiquitin.Treatment with CXCL12 promoted ubiquitination of CXCR4, compared tovehicle treated cells, similar to what we have previously observed (FIG.12C). CXCL12₁ also promoted CXCR4 ubiquitination, although not to thesame degree as CXCL12 (FIG. 12C). In contrast, dimeric SDF-1α failed topromote CXCR4 ubiquitination, consistent with its inability to promoteCXCR4 degradation (FIG. 12C).

We have previously shown that AIP4-dependent ubiquitination of CXCR4 isdependent upon phosphorylation of serine residues 324 and 325 in theC-tail. To examine the effect of CXCL12 variants on serinephosphorylation, we performed confocal immunofluorescence microscopyusing an anti-mouse CXCR4 antibody that selectively recognizes pS324 andpS325. HeLa cells transfected with HA-CXCR4-YFP were treated with 80ng/ml vehicle, CXCL12, CXCL12₁, and CXCL12₂ for 15 min. Vehicle treatedcells exhibited very little staining consistent with CXCR4 beingunphosphorylated under basal conditions CXCR4 (FIG. 12D).

In contrast, treatment with CXCL12 or CXCL12₁ produces strong stainingsuggesting that CXCR4 is robustly phosphorylated on serine residues 324and 325 (FIGS. 12E, 12F). However, CXCL12₂ treatment resulted in weakstaining similar to vehicle indicating that CXCL12₂ does not promoteCXCR4 phosphorylation on serine residues 324 and 325.

In summary, CXCL12₁ is able to promote robust internalization,phosphorylation and ubiquitination of CXCR4 and hence its degradation.In contrast, CXCL12₂ does not efficiently promote internalization,phosphorylation and ubiquitination of CXCR4 and thus does not promoteCXCR4 degradation.

CXCL12₁ enhances apoptosis in acute myeloid leukemia cells. Lastly, webegan to explore the therapeutic potential of CXCL12₁. We previouslyshowed that CXCL12_(WT) could mitigate colorectal and melanomametastasis in vivo, but CXCL12₂ was a more potent inhibitor. Kremer etal. recently discovered an unexpected role for CXCL12 as an inducer ofapoptosis in acute myeloid leukemia cell lines and clinical isolates. Todetermine the relevant CXCL12 variant, we exposed KG1a leukemia cells toCXCL12 variants for 16-18 h and then stained for annexin V. BothCXCL12_(WT) and CXCL12₁ treatment produced a robust dose-dependentincrease in annexin V with EC₅₀ values of 1.75±0.6 and 2.38±0.6 nM,respectively (FIG. 12H). In contrast, CXCL12₂ resulted in a small butsignificant increase in annexin V. To confirm that the annexin Vstaining was reporting on apoptosis we also measured the presence ofcleaved PARP. As expected, all three variants enhanced the cleavage ofPARP but the CXCL12_(WT) and CXCL12₁ were significantly greater thanCXCL12₂ (FIG. 12I). CXCL12-mediated apoptosis is unaffected by theG_(i)-type protein inhibitor pertussis toxin. This suggests apoptosis isdependent on β-arrestin-2 signaling, which is consistent with the weakrecruitment mediated by CXCL12₂.

Radioligand Displacement.

Commercial membrane preparations of cells stably expressing CXCR4 orCXCR7 were purchased (Millipore). Five micrograms of protein per pointwas incubated for 90 min on ice in binding buffer with 0.03 nM of¹²⁵I-CXCL12 as a tracer and increasing concentrations of competitor in afinal 40 μL volume. Bound radioactivity was separated from free ligandby filtration, and receptor-bound radioactivity was quantified bygamma-radiation counting (Perkin-ECXCL121er Life and AnalyticalSciences). Isolated membrane binding experiments were carried out induplicate.

THP-1 Calcium Response.

THP-1 monocyte cells were washed twice and resuspended in 96-well formatat 2×10⁵ cells/well in assay buffer: Hanks buffered saline solution(HBSS), 20 mM HEPES (pH 7.4), 0.1% (w/v) BSA, and FLIPR Calcium4 dye(Molecular Devices) and then incubated for 1 h at 37° C., 5% CO₂.Fluorescence was measured at 37° C. using a FlexStation3 MicroplateReader (Molecular Devices) with excitation and emission wavelengths at485 nm and 515 nm, respectively. Chemokines were resuspended at theindicated concentrations and added to the cells following a 20 sbaseline fluorescence measurement. Percent calcium flux was calculatedfrom the maximum fluorescence minus the minimum fluorescence as apercent of baseline. EC₅₀ values were determined by non-linear fittingto a four parameter logistic function.

THP-1 Chemotaxis.

Chemotaxis experiments were performed using Tranwell (5 mm pore size;Costar) in 24-well format. The indicated concentrations of CXCL12₁ wereresuspended in RPMI 1640 containing 0.2% (w/v) BSA and added to thelower chamber. THP-1 cells were washed twice and 5×10⁵ cells were addedto the upper chamber. Plates were incubated for 3 h at 37° C., 5% CO₂.Following incubation, Transwell membranes were removed and cells thatmigrated into the lower chamber were counted using a TC-10 AutomatedCell Counter (BioRad) and hemocytometer. The chemotactic index wascalculated as the difference in migrated cell number between thoseexposed to chemokine and the vehicle control.

NALM6 Chemotaxis.

NALM6 cells were grown in RPMI-1640 supplemented with 10% FBS, 100 U/mLpenicillin-streptomycin, 2 mM glutamine, 50 μM α-mercaptoethanol,non-essential amino acids, 1 mM Na-pyruvate and 25 mM HEPES buffer (pH7.3). Chemotaxis assays were performed in triplicate in 48-well Boydenchambers (NeuroProbe), using 5 μm pore-sized polyvinylpyrrolidone-freepolycarbonate membranes. Chemotaxis medium (RPMI-1640, 25 mM HEPESsupplemented with 1% FBS) alone or chemotaxis medium containingincreasing concentrations of CXCL12 variants was added to the lowerwells. Cells (1×10⁵ per well) resuspended in chemotaxis medium wereadded to the upper well and incubated for 90 min at 37° C. in 5% CO₂atmosphere. Cells were removed from the upper part of the membrane witha rubber policeman. Cells attached to the lower side of the membranewere fixed and stained as described. Migrated cells were counted in fiverandomly selected fields of 1000-fold magnification.

MiaPaCa2 Chemotaxis.

Chemotactic migration of pancreatic cancer MiaPaCa2 cells was performedas previously described using Transwell plates coated with 15 ug/mlcollagen. Briefly, MiaPaCa2 cells were serum starved for 2 hours, liftedusing enzyme-free dissociation buffer, washed and then counted using ahemocytometer. 100,000 cells in 10 uL of serum free media were platedinto the top chamber of each Transwell. The bottom chamber of eachTranswell contained each stimulant in 500 uL serum free media. MiaPaCa2cells were allowed to migrate for 6 hours, after which the cellsremaining on the top of the chamber were swabbed out. Plates were thenfixed in 4% paraformaldehyde and stained with DAPI. Migrated cells werevisualized and counted by fluorescence microscopy, with 5 representativehigh-powered fields taken per well.

β-arrestin-2 recruitment.

β-arrestin-2 recruitment was measured using an intermolecular BRET assayperformed as described previously. HEK293E cells were cotransfected with1 mg of CXCR4-eYFP construct with 0.05 mg of β-arrestin-2-RLuc (a giftfrom Michel Bouvier, University of Montreal, Montreal, QC, Canada). For[acceptor]/[donor] titrations 0.05 mg of β-arrestin-2-RLuc werecotransfected with increasing amounts of the CXCR4-eYFP construct. Alltransfections were completed to 2 mg/well with empty vector. Followingovernight culture, transiently transfected cells were seeded in 96-well,white, clear-bottom microplates (View-Plate, Perkin-ECXCL121er Life andAnalytical Sciences), coated with poly-D-lysine, and left in culture for24 h. Cells were washed once with PBS, and the RLuc substratecoelenterazine-H (NanoLight Technology) was added at a finalconcentration of 5 mM to BRET buffer (PBS, 0.5 mM MgCl₂, 0.1% w/vglucose). β-arrestin recruitment was measured 30 min after ligandaddition. The values were corrected to BRETnet by subtracting thebackground BRET signal obtained in cells transfected with RLuc constructalone.

Agarose Microdroplet Assay.

The agarose microdroplet assay was performed to determine U-937 cellularmigration, as previously described. 0.5×10⁶ cells/ml U-937 target cellswere harvested and washed in HBSS. Cells were spun at 2,000 rpm and weretransferred to a graduated 15 ml glass conical tube. The cellconcentration was adjusted in agarose medium, prepared from 2% lowmelting temperature Seaplaque agarose and medium containing 15% FBS,using a 1:4 volume-to-volume dilution. A 1 μl agarose droplet containing1×10⁵ target cells was placed in the center of each well of a 96-wellflat-bottom tissue culture plate, in triplicate, using a gastight 0.05ml Hamilton syringe (Hamilton Company, Reno, Nev.). Droplets wereallowed to harden at 4° C. for 20 min. 200 μl chilled test media wasapplied to each well. Test medium consisted of serum-free medium, 25%FBS, and the indicated CXCL12 concentrations. The plate was incubatedfor 18-24 h at 37° C. and 5% CO₂. Following incubation, the radius ofeach droplet was determined and target cell migration was measured atfour directional points 90° from one another, using an inverted lightmicroscope equipped with a gridded eyepiece, at 40× total magnification.Percent inhibition of each sample was quantified. The plate wasincubated for an additional 24 h to measure recovery, and viability wasdetermined by trypan blue exclusion.

NMR Structure Determination.

All NMR spectra were acquired on a Bruker DRX 600 MHz spectrometerequipped with a ¹H, ¹⁵N, ¹³C TXI cryoprobe at 298 K. Experiments wereperformed in a solution containing 25 mM deuterated MES (pH 6.8), 10%(v/v) D₂O, and 0.02% (w/v) NaN₃. NOE distance restraints were obtainedfrom 3D ¹⁵N-edited NOESY-HSQC, aliphatic ³C-edited NOESY-HSQC, andaromatic ³C-edited NOESY-HSQC spectra (τ_(mix)=80 ms) collected on both[U-¹⁵N,¹³C]-CXCL12₁ saturated with CXCR4₁₋₃₈ and [U-¹⁵N,¹³C]-CXCR₁₋₃₈saturated with CXCL12₁. Intermolecular NOEs were obtained from a 3DF1-¹³C/¹⁵N-filtered/F3-¹³C-edited NOESY-HSQC (τ_(mix)=120 ms) collectedon both [U-¹⁵N,¹³C]-CXCL12₁ saturated with CXCR4₁₋₃₈ and[U-¹⁵N,¹³C]-CXCR₁₋₃₈ saturated with CXCL12₁. In addition to NOEs,backbone φ/ψ dihedral angle restraints were derived from ¹H^(N), ¹H^(α),¹³C^(α), ¹³C^(β), ¹³C′, and ¹⁵N chemical shift data using TALOS+. Bothdistance and dihedral restraints were used to generate initial NOEassignments and preliminary structures via the NOEASSIGN module ofCYANA. Complete structure determination was undertaken as an iterativeprocess of correcting/assigning NOEs and running structure calculationswith CYANA. The 20 CYANA conformers with the lowest target function werefurther refined by a molecular dynamics protocol in explicit solventusing XPLOR-NIH.

2D NMR Characterization.

All NMR spectra were acquired on a Bruker DRX 600 MHz spectrometerequipped with H, ¹⁵N, ¹³C TXI cryoprobe at 298 K. Experiments wereperformed in a solution containing 25 mM deuterated MES (pH 6.8), 10%(v/v) D₂O, and 0.02% (w/v) NaN₃. Heteronuclear NOE experiments werecollected on 250 μM [U-¹⁵N]-CXCR4₁₋₃₈ in the absence and presence of 500μM CXCL12₁. ¹⁵N-HSQC spectra were collected to monitor the interactionof 200 μM [U-¹⁵N]-CXCR4₁₋₃₈ was titrated with 0, 100, 200, 300, 400, and500 μM CXCL12₂. ¹⁵N-HSQC spectra were collected to monitor theinteraction of 250 μM [U-¹⁵N]-CXCR41-38 was titrated with 0, 62.5, 125,187.5, 250, 375 and 500 μM CXCL12₁.

Calcium Response of CXCR4 Mutants.

Cell culture, transfection and calcium flux assay of Chinese hamsterovary K1 (CHO-K1) cells was performed as previously described.

Cell Lines, Antibodies and Other Reagents.

HEK (Human embryonic kidney) 293 (Microbix, Toronto, Canada) and HeLa(American Type Culture Collection) cells were maintained in Dulbecco'smodified Eagles medium (DMEM; Hyclone) supplemented with 10% fetalbovine serum (FBS; HyClone Laboratories, Logan, Utah). The PE-conjugatedCXCR4 (CD184) isotype control antibodies, and rat anti-CXCR4 (2B11) andwere from BD Biosciences (San Jose, Calif.). The anti-actin antibody wasfrom MP Biomedicals (Aurora, Ohio). The Alexa-Fluor 568-conjugated goatanti-rabbit antibody was from Molecular Probes (Eugene, Oreg.). Theanti-FLAG M2 horseradish peroxidase conjugated monoclonal antibody wasfrom Sigma (St. Louis, Mo.). The anti-HA polyclonal and monoclonalantibodies were from Covance (Berkeley, Calif.). CXCL12 was fromPeproTech (Rockyhill, N.J.).

CXCR4 Degradation Assay.

HeLa cells grown on 6-cm dishes were washed once and incubated with DMEMcontaining 10% FBS and 50 μg/ml cyclohexamide for 15 min at 37° C. Cellswere then incubated in the same medium containing vehicle (PBScontaining 0.5% bovine serum albumin [BSA]), 80 ng/ml CXCL12_(WT),CXCL12₁, or CXCL12₂ for 3 h. Cells were washed once and collected in 300μl 2× sample buffer. Receptor levels were determined by SDS-PAGEfollowed by immunoblotting using an anti-CXCR4 antibody. Blots werestripped and reprobed for actin to assess loading. Receptor levelsnormalized to actin levels were determined by densitometric analysis.

CXCR4 Internalization Assay.

HeLa cells grown on 6-cm dishes were washed twice with PBS and detachedfrom the surface of the plate by incubating with 350 μl Cellstrippercell dissociation solution (Mediatech, VA) for 10 min at 37° C. Cellswere collected in 4 ml PBS containing 0.1% BSA (Media Tech, VA) bycentrifugation. Cell pellets were re-suspended in 1.5 ml PBS-0.1% BSAand 5×10⁵ cells were transferred to a fresh 5 ml polystyrene roundbottom tube (BD falcon) and washed once with PBS+0.1% BSA andre-suspended in 250 μl PBS-0.1% BSA. Cells were incubated at 37° C. for15 min and then treated with vehicle, 80 ng/ml CXCL12_(WT), CXCL12₁, orCXCL12₂ for 20 min at 37° C. Following treatment, 4 mL cold PBS wasadded to each tube, cells were collected by centrifugation and thenfixed by re-suspending the pellet in 500 μl 4% paraformaldehyde (made inPBS) and incubating for 15 min at 37° C. Cells were collected bycentrifugation and washed once with 4 mL PBS and then twice withPBS-0.1% BSA. Cells were stained with PE-conjugated anti-CXCR4 orisotype control antibodies by re-suspending the pellet in 100 μlPBS-0.1% BSA (supplemented with 5% normal goat serum) containinganti-CXCR4 antibody (1:100 dilution) and incubating for 1 hour at roomtemperature in the dark. Following staining, cells were washed twicewith 4 mL PBS-0.1% BSA. Finally, cells were re-suspended in 300 μlPBS-0.1% BSA and kept in the dark until the analysis was performed.CXCR4 surface expression was analyzed by flow cytometry (FACS-CANTO;Becton Dickinson) and analysis was performed using FlowJo v.9.3software.

CXCR4 Ubiquitination Assays.

HEK293 cells stably expressing HA-CXCR4 grown on 10-cm dishes weretransfected with 3 μg FLAG-ubiquitin. The next day, cells were passagedonto 6-cm dishes and allowed to grow for an additional 24 h. Thefollowing day, cells were incubated in DMEM containing 20 mM HEPES for 6h and then treated with vehicle (PBS, 0.1% BSA), 80 ng/ml CXCL12_(WT),CXCL12₁, or CXCL12₂ for 30 min. Cells were then washed once on ice withcold PBS, and collected in 1 ml lysis buffer [50 mM Tris-Cl, pH 7.4, 150mM NaCl, 5 mM EDTA, 0.5% (w/v) sodium deoxycholate, 1% (v/v) NP-40, 0.1%(w/v) SDS, 20 mM N-ethyCXCL121aleimide (NEM), and 10 μg/ml each ofleupeptin, aprotinin, and pepstatin A]. Samples were transferred intomicrocentrifuge tubes and placed at 4° C. for 30 min, followed bysonification and centrifugation to pellet cellular debris. Clarifiedcell lysates were incubated with an anti-HA polyclonal and isotypecontrol antibodies and the immunoprecipitates were analyzed by SDS-PAGEfollowed by immunoblotting using an anti-FLAG antibody conjugated toHRP.

Confocal Microscopy.

HeLa cells transiently transfected with HA-CXCR4-YFP were passaged ontopoly-L-lysine coated coverslips and grown for 24 h. The next day, cellswere washed once with warm DMEM containing 20 mM HEPES, pH 7.5, andincubated in the same medium for 3-4 h at 37° C. Cells were treated with80 ng/ml CXCL12_(WT), CXCL12₁, or CXCL12₂ and vehicle for 30 min. Cellswere then fixed with PBS containing 3.7% paraformaldehyde, followed bypermeabilization with 0.05% (w/v) saponin for 10 min, similar to aprotocol we have described previously. Cells were then incubated with 1%goat serum in 0.05% saponin-PBS for 30 min at 37° C., followed bystaining with anti-mouse monoclonal antibody that recognizes duallyphosphorylated CXCR4 on serine residues 324 and 325 (clone 5E11) (1:50dilution) for 1 h at 37° C. Cells were washed five times with 0.05%saponin-PBS, followed by incubating with Alexa-Fluor 568-conjugatedanti-rabbit antibody for 30 min at 37° C. (1:200 dilution). Finally,cells were washed five times with 0.05% saponin-PBS and mounted ontoglass slides using mounting media containing4,6-diamidino-2-phenylindole (Vectashield mounting media with DAPI).Samples were analyzed using an LSM 510 laser scanning confocalmicroscope (Carl Zeiss, Thornwood, N.Y.) equipped with a Plan-Apo63×/1.4 oil lens objective. Images were acquired using a 1.4-megapixelcooled extended spectra range RGB digital camera set at 512×512resolution. Acquired images were analyzed using ImageJ, version 1.41osoftware (National Institutes of Health, Bethesda, Md.) and AdobePhotoshop (CS4).

Apoptosis Assays.

KG1a (human acute myelogenous leukemia) cells were assayed forCXCL12-dependent apoptosis as previously described. Briefly, CXCR4expression on KG1a cells was achieved via transient transfection with aplasmid encoding a CXCR4-YFP fluorescent fusion protein. These cellswere treated with the indicated concentration of each CXCL12 variant andcultured for 16-18 h prior to measuring apoptosis. Apoptosis was assayedby staining cells with APC-conjugated annexin V to detect cell-surfacephosphatidyl serine (BD Biosciences) or Alexaflour-647-conjugatedantibody to detect cleaved PARP (BD Biosciences), and the percentage ofYFP+ cells undergoing apoptosis was determined by flow cytometry.

Statistical Analysis and Final Figures.

Data were analyzed by one-way analysis of variance (ANOVA) usingGraphPad Prism 4.0 (GraphPad Software).

Example 3. Structural Basis for Balanced CXCR4 Signaling by aConstitutively-Monomeric CXCL12 Variant

In this example, the inventors show that an engineered CXCL12locked-monomer functions as a balanced agonist with enhanced G-proteinand arrestin signaling. The balanced agonist alone directs the fate ofCXCR4 by promoting phosphorylation, ubiquitination, internalization anddegradation. These modifications originate at the plasma membranesuggesting that balanced and biased agonists stabilize unique receptorconformations. We then solved the NMR complex structure of monomericCXCL12 and the CXCR4 N-terminus and observed a monomer-specificinteraction that is required for receptor activation. A model of thefull-length 1:1 receptor complex from component NMR and crystallographicstructures, coupled with experimental validation, reveals a contiguousinterface for rational ligand design.

Here we used a constitutively-monomeric CXCL12 variant (CXCL12₁) toexplore balanced CXCR4 signaling. After confirming that the CXCL12₁activates both G protein and arrestin pathways, the fate of activatedcell-surface CXCR4 was tracked following activation with both thebalanced or G protein biased agonist. Only the CXCL12 monomer promotesCXCR4 phosphorylation, ubiquitination, and degradation. We thendetermined the structure of CXCL12₁ bound to the N-terminal domain ofCXCR4 (CXCR4₁₋₃₈). Apolar residues near the CXCR4 N-terminus dock into acleft that is inaccessible in the dimer, and this monomer-specificinteraction is essential for full receptor activation. Combination ofour CXCL2:CXCR4₁₋₃₈ complex NMR structure and the CXCR4-inhibitorcrystal structure permitted modeling of an intact 1:1 complex. Our modelextends the conceptually useful “two-site” model and suggests thatreceptor activation involves the formation of an extensiveprotein-protein interface encompassing nearly half of the CXCL12surface.

CXCL12₁ is a Balanced CXCR4 Agonist with Enhanced G Protein and ArrestinSignaling.

At low concentrations CXCL12 is hypothesized to interact with CXCR4 as amonomer to promote cellular migration; as local chemokine concentrationsincrease the dimeric form predominates and induces non-migratorycellular idling signaling. Similarly, peptides derived from the CXCR4N-terminus bind CXCL12 in a 1:1 stoichiometry but also promote formationof a 2:2 complex at higher concentrations. Binding of CXCR4 peptideswith specific tyrosine sulfation patterns can even allostericallymodulate chemokine dimerization. To simplify this complex equilibrium weengineered a disulfide-constrained CXCL12 variant (CXCL12₁) that remainsstrictly monomeric at millimolar concentrations. To test if CXCL12₁ isfunctionally equivalent to the monomeric form of CXCL12_(WT), wecompared their relative ability to recognize and activate CXCR4.Radioligand displacement on CXCR4 cell-membrane preparationsdemonstrated similar affinities for CXCL12_(WT) and CXCL12₁ with K_(d)values of 1.4±1.5 nM and 0.97±1.5 nM, respectively (FIG. 18A). We nextestablished the activity of CXCL12₁ as a CXCR4 agonist by measuring itsability to mobilize intracellular calcium, a sensitive indicator of Gprotein activation. CXCL12₁ produced a robust, dose-dependent calciumflux response equivalent to CXCL12_(WT) (FIG. 18B). Chemokines inducecellular migration over a narrow concentration range when measured usinga Boyden chamber or similar apparatus. The monomer-dimer equilibriummight contribute to CXCL12's narrow bell-shaped chemotactic profile,with the loss of migration at higher concentrations attributed to theformation of inhibitory dimeric complexes. Whereas a disulfide-locked,constitutively dimeric CXCL12 variant (CXCL12₂) does not promotechemotaxis at any concentration, a preferentially monomeric CXCL12variant produces a bell-shaped response over an extended concentrationrange. Because CXCL12₁ is strictly monomeric, we hypothesized it maygenerate a sigmoidal, rather than a biphasic, migratory response. Thechemotaxis of NACXCL1216 pre-B cells and MiaPaCa2 pancreatic cancercells was tested using Boyden and Transwell migration chambers,respectively (FIGS. 18C, 18D). In both cases CXCL12₁ extended theeffective dose at least 10-fold relative to CXCL12_(WT) but,nonetheless, signaled migratory arrest at the highest concentrations. Toassess how CXCR4 activation was interpreted in the presence of othermigratory signals, U-937 leukemia cells confined to an agarose dropletwere incubated in serum-containing media with increasing CXCL12concentrations. Whereas CXCL12_(WT) inhibited U-937 cell migration atnearly all concentrations with maximal inhibition of 40.1±18%, CXCL12₁significantly enhanced migration, generating a bell-shaped profile, atall tested concentrations (FIG. 18E).

Chemotaxis is dependent upon recruitment of β-arrestin to CXCR4, whichthen induces lamellipodia formation and filamentous-actinpolymerization. We recently demonstrated that monomeric CXCL12 isprimarily responsible for β-arrestin-2 mobilization and subsequentinternalization. We next performed dose-dependent BRET analysis to testif the CXCL12₁'s enhanced chemotactic profile was reflected inβ-arrestin signaling. At low concentrations, CXCL12_(WT) and CXCL12₁both recruited β-arrestin to CXCR4 with similar potency and efficacy(FIG. 18G). However, as concentrations were increased to 10 and 100 μM,CXCL12_(WT) induced a bimodal stimulation of β-arrestin reminiscent ofits chemotactic dose response. In contrast, CXCL12₁ exhibits a sigmoidalresponse over the tested concentration range. Enhanced arrestinrecruitment suggests that CXCL12₁ binding directly modifies thereceptor's intracellular conformation which may effect other aspects ofthe receptor's fate.

CXCL12₁ Enhances Apoptosis in Acute Myeloid Leukemia Cells.

Lastly, we began to explore the therapeutic potential of CXCL12₁. Wepreviously showed that CXCL12_(WT) could mitigate colorectal andmelanoma metastasis in vivo, but CXCL12₂ was a more potent inhibitor.Kremer et al. recently discovered an unexpected role for CXCL12 as aninducer of apoptosis in acute myeloid leukemia (AML) cell lines andclinical isolates. To identify the relevant CXCL12 variant, we exposedKG1a leukemia cells to CXCL12 variants for 16-18 h and then stained forannexin V. Both CXCL12_(WT) and CXCL12₁ treatment produced a robustdose-dependent increase in annexin V with EC₅₀ values of 1.75±0.6 and2.38±0.6 nM, respectively (FIG. 19G). In contrast, CXCL12₂ resulted in asmall but significant increase in annexin V. To confirm that the annexinV staining was reporting on apoptosis we also measured the presence ofcleaved PARP. As expected, all three variants enhanced the cleavage ofPARP but the CXCL12_(WT) and CXCL12₁ were significantly greater thanCXCL12₂ (FIG. 19H). CXCL12-mediated apoptosis is unaffected by theGi-type protein inhibitor pertussis toxin, suggesting that AML apoptosisis primarily dependent on β-arrestin signaling and thereforeunresponsive to CXCL12₂. Counterintuitively, AML cells possess highsurvival rates in the CXCL12-rich bone marrow microenvironment. Our datasuggests the survival may result from concentration-dependent CXCL12dimerization.

Monomeric CXCL12 Mediates CXCR4 Degradation, Phosphorylation andUbiquitination.

In addition to their classic role of promoting internalization anddesensitization, arrestins sometimes function as scaffolds for secondarysignaling such as ubiquitination. To investigate the effect of ligandoligomeric state on receptor trafficking, we initially examined CXCR4lysosomal targeting and degradation. HeLa cells were treated for 3 hwith 80 ng/ml CXCL12_(WT), CXCL12₁, or CXCL12₂, and receptor levels weredetected by immunoblot analysis. Both CXCL12_(WT) and CXCL12₁ promotedapproximately 60% CXCR4 degradation in contrast to 15% degradation byCXCL12₂, (FIG. 19A). Internalization is a prerequisite for degradation;therefore, we next examined the ability of these ligands to promoteCXCR4 internalization. HeLa cells were treated with CXCL12_(WT),CXCL12₁, and CXCL12₂ for 20 min followed by FACS measurement of CXCR4surface expression. As shown in FIG. 19B, CXCL12₁ and CXCL12_(WT)promoted similar levels of CXCR4 internalization. However, the nominalinternalization promoted by CXCL12₂ was consistent with inefficientdegradation (FIGS. 19A, 19B) and our previous observations.

Lysosomal targeting and degradation of CXCR4 is dependent uponC-terminal modification by the E3 ubiquitin ligase AIP4. We nextexamined the effect of CXCL12_(WT), CXCL12₁, and CXCL12₂, on CXCR4ubiquitination. HEK293 cells stably expressing HA-CXCR4 and transfectedwith FLAG-ubiquitin were treated with each CXCL12 variant for 30 min.HA-CXCR4 was immunoprecipitated and the ubiquitinated receptor wasdetected by immunoblotting for the FLAG epitope. Treatment withCXCL12_(WT) prompted ubiquitin. CXCL12₁ also promoted CXCR4ubiquitination, although not to the same degree as CXCL12_(WT) (FIGS.19C, 19D). Consistent with its inability to stimulate receptorinternalization and degradation, CXCL12₂, failed to promote CXCR4ubiquitination (FIG. 19C, 19D).

We have previously shown that AIP4-dependent ubiquitination of CXCR4 isdependent upon phosphorylation of Ser324 and Ser325. Ser324/Ser325 arephosphorylated at the plasma membrane by protein kinase Cδ and GRK6. Toexamine the effect of CXCL12 variants on serine phosphorylation, weperformed confocal immunofluorescence microscopy using an anti-mouseCXCR4 antibody that selectively recognizes pSer324/325. HeLa cellstransfected with HA-CXCR4-YFP were treated with 80 ng/ml vehicle,CXCL12_(WT), CXCL12₁, or CXCL12₂ for 15 min. Vehicle treated cellsexhibited very little staining consistent with CXCR4 beingunphosphorylated under basal conditions (FIG. 19E). Addition ofCXCL12_(WT) or CXCL12₁ produced strong staining indicating robust CXCR4C-terminal phosphorylation (FIGS. 19F, 19G). In striking contrast,CXCL12₂ treatment resulted in weak staining similar to vehicle (FIG.19H). Taken together, our results indicate that CXCR4 ligand bindingdirectly modifies the receptor's intracellular conformation, yieldingthe respective balanced or biased response.

The Structure of CXCL12₁ in Complex with CXCR4-38.

We next used nuclear magnetic resonance (NMR) spectroscopy to explorethe structural mechanisms that translate CXCL12₁ binding into balancedagonism. Chemokine signaling is initiated by formation of an extensiveprotein-protein interface segregated into two distinct regions. First,the receptor N-terminus wraps around the folded chemokine domain andcontributes most of the binding energy (site 1). Subsequent docking ofthe flexible chemokine N-terminus into a pocket within the transmembranedomain activates receptor signaling (site 2). Using a 38 amino acidCXCR4 N-terminal peptide (CXCR4₁₋₃₈), we previously showed thatincreasing CXCL12_(WT) concentrations caused [U-¹⁵N]-CXCR4₁₋₃₈resonances to shift in curved trajectories suggesting the simultaneouspresence of both a 1:1 and 2:2 complexes. Here we used HSQC titrationsto probe the 1:1 and 2:2 interfaces. Titrating CXCL12₁ or CXCL12₂ into[U-¹⁵N]-CXCR4₁₋₃₈ produced chemical shift perturbations consistent withunique chemical environments for CXCR4 residues 1-13 (FIG. 22A). Incontrast to previously published spectra of CXCL12_(WT), all peaks inthe N-terminus were visible throughout the titrations and traversedlinear paths. Concatenation of the linear trajectories recreates thecomplicated chemical shifts induced by CXCL12_(WT) and underscores thephysiologic validity of both the CXCL12₁ and CXCL12₂ variants (FIG.22A). To assess the rigidity of CXCR4₁₋₃₈ upon chemokine binding wemeasured ¹H-¹⁵N heteronuclear NOE values, which reflect the backboneflexibility for each residue on picosecond to nanosecond timescales.When bound to CXCL12₂, CXCR4₁₋₃₈ residues 5-10 remain relatively dynamicand imply a weak, transient interaction. CXCL12₁ stabilizes the highfrequency motions of CXCR4₁₋₃₈ residues 5-10 (FIG. 22B).

To understand how the CXCR4 N-terminus recognizes CXCL12₁, we determinedthe complex structure by NMR. A suite of triple-resonance experimentswere acquired on samples of [U-¹³C,¹⁵N]-CXCL12₁ saturated with CXCR4₁₋₃₈and [U-¹³C,¹⁵N]-CXCR4₁₋₃₈ saturated with CXCL12₁ to assign the carbon,nitrogen, and hydrogen resonances. Next, a series of 3D nuclearOverhauser effect (NOE) spectroscopy (NOESY) experiments were collectedto identify the position of neighboring hydrogen atoms within a 5 Ådistance. As expected, CXCL12₁ adopts the canonical chemokine foldcomprising a flexible N-terminus, followed by the N-loop, athree-stranded antiparallel β-sheet, and a C-terminal α-helix (FIGS.20A-C). CXCL12₁ was designed to mimic the conformation of PDB 1SDF, aCXCL12 solution NMR structure determined in sodium acetate buffer at pH4.9. In this structure the α-helix maintains a 550 relative to theβ-sheet, which would be sterically incompatible with dimerization. TheCXCL12₁ α-helix is 65° relative to the β-sheet and aligns to PDB 1SDFwith a backbone RMSD of 1.6 Å(FIG. 23).

Determination of the CXCR4₁₋₃₈ contact surface requiredF1-¹³C-filtered/F3-¹³C-edited NOESY-HSQC experiments to unambiguouslyidentify intermolecular constraints. Intermolecular NOEs, indicative ofa stable interaction on the millisecond timescale, were observed fromresidues 4-27 along the peptide (FIGS. 20A-C). Overall, CXCR4₁₋₃₈ adoptsa random coil architecture with a short β-strand from Tyr7-Ser9. Theposition of CXCR-38 overlaps well with the previously published chemicalshift perturbations (FIGS. 20A-C). The ProCys motif (CXCR4 Pro27 andCys28) is nearly conserved across all chemokine receptors and delineatesthe flexible N-terminus from the transmembrane portion. Here we observedintermolecular NOEs from both the CXCL12₁ N-loop and helix to CXCR4Lys25, Glu26, and Ala28. This constrains CXCR4 Pro27 between the helixand N-loop, marking the point where our NMR data intersects with therecent CXCR4 crystal structure. The Pro27 Ca is translated an average8.6 Å toward the N-loop compared to the 2:2 complex. Subsequently, thepocket formed by the N-loop and β3 strand, known as the chemokine cleft,is slightly distorted. CXCL12₁ residues Glu15-His17 shift toward the β3strand to accommodate the peptide. CXCL12₁ Asn46 puckers away from thepocket allowing Arg47, and to a lesser extent Ans45, to interact withthe CXCR4 Tyr21 hydroxyl. The C^(ζ) and C^(α) atoms of Tyr21 aretranslated an average of 7.0 Å and 5.3 Å, respectively, compared to the2:2 complex (FIG. 20D). In this position the Tyr21 hydrophobic contactsappear to be primarily satisfied by Val49 and the methylene of Glu15.Upon sulfation, it's reasonable to predict that Lys47, Asn45 or His17maintain sTyr21 electrostatic interactions with negligible pocketrearrangement.

The 1:1 and 2:2 complex interfaces sharply diverge at residuesN-terminal of CXCR4 Tyr12. In the 2:2 complex, CXCR4 Tyr12 makeselectrostatic contacts with Lys27 of one CXCL12₂ protomer and His25 ofthe other. Upon sulfation, sTyr12 preferentially forms salt bridges withLys27 from a single protomer. Tyr7 also makes contacts with the otherCXCL12₂ subunit by burying into a pocket formed by Val23 and Arg20. Inthe 1:1 complex Tyr12 buries into a deep cleft formed by Pro10, Lys27,Leu29, and Val 39 that possesses no obvious electrostatic or chargeinteractions for a sulfated tyrosine. Asp10 and Asn11 of CXCR4₁₋₃₈ thenturn to place Tyr7 in close proximity to Tyr12. The Tyr7 hydroxyl ispositioned toward His25 and Lys27, but productive contacts are notclear. CXCR4₁₋₃₈ residues Tyr7-Ser9 hydrogen bond with CXCL12₁Ile28-Asn30 to form a four-stranded β-sheet (FIG. 20E; FIG. 24). Theposition of Tyr7 enables Ile6 to bury into a hydrophobic cleftsurrounded by Leu26, Tyr61, and Ala65 (FIG. 20F). Ile4 packs further upthe helix against CXCR4 Ile6 and CXCL12₁ Leu26, Trp57, and Tyr61. Theinteraction of hydrophobic CXCR4 residues with the CXCL12 helix isconsistent with previous NMR titration studies of CXCR4₁₋₃₈ andcross-saturation NMR experiments with full-length CXCR4. Sulfation ofTyr7 reduces the binding affinity of a CXCR4 Ile4-Asp10 heptapeptidetwo-fold. The reduced affinity may result from a sulfated Tyr7 strainingto interact with Lys27 and displacing the isoleucines from theirhydrophobic cleft.

The CXCR4 N-Terminus is Critical for Chemokine Recognition andActivation.

The CXCL12₁:CXCR4₁₋₃₈ complex contains a unique interface for residues1-12 that is incompatible with CXC-type chemokine dimerization (FIGS.20G, 20H). To assess the contributions of CXCR4 Ile4 and Ile6, wedesigned a series of CXCR4 mutants and monitored binding affinity andcalcium flux dose responses. Ile4 and Ile6 were simultaneously mutatedto either alanine or glutamic acid residues in FLAG-tagged CXCR4 (FIG.25). Whereas alanine substitution had no effect on binding, the affinityof CXCL12_(WT) and CXCL12₁ for glutamate mutants was reduced 30- and90-fold, respectively (FIG. 20I). Similarly, receptor activation,monitored by the dose-dependent calcium response in CHO-KI cells, wasreduced 10-fold upon mutagenesis to glutamatic acid (FIG. 20J).

Structure-Guided Modeling of the Full-Length 1:1 Receptor Complex.

No CXCL12:CXCR4 complex structure exists to date; in part because of theligand's dimer equilibrium and the receptor's inherent conformationalflexibility. Recently, Handel and colleagues proposed that both site 1and site 2 interactions require only a single protomer of the CXCR4dimer for full-agonist activation. Combining our CXCL12₁:CXCR4₁₋₃₈ NMRstructure with the previously-published CXCR4 crystal structures, wewere uniquely positioned to model the complete full-agonist complex(FIG. 21A). The docking of CXCL12₁ to CXCR4 proceeded in five steps. Wefirst docked the CXCL12₁ N-terminal peptide (residues 1-8; KPVSLSYR)into the orthosteric site of CXCR4 (PDB 30DU:A residues 29-301) usingthe FlexPepDock ab initio protocol. In the second step, the N-terminalpeptide model from the first step and CXCR4 Pro27 (anchored by adisulfide between Cys28-Cys274) were used to roughly guide the placementof the CXCL12₁:CXCR4₁₋₃₈ NMR structure. In the third step, we optimizedthe model using the RosettaRelax protocol, and connected the structureddomain of CXCR4 to the CXCR4₁₋₃₈ fragment using the Rosetta loopmodeling protocol. Finally, CXCL12₁ residues 1-8 were re-docked toadjust to the relaxed complex using FlexPepDock ab initio.

Next, we inspected the model for known structure activity relationships.Deletion studies by Crump established a significant role for CXCL12residues 1-5 in receptor recognition and activation, but showed thatSer6 and Tyr7 were not essential complex formation. In our model thechemokine N-terminus participates in both polar and apolar contacts toCXCR4 residues previously identified as critical in mutagenic calciumflux experiments. CXCL12 Lys1 sits at the base of the pocket andinteracts with CXCR4 Asp97^(2.63) and Glu288^(7.39) (FIG. 21B), whichboth contribute to ligand binding and are essential for receptoractivation. CXCR4 His281^(7.32) forms a polar contact with the carbonylof CXCL12 Pro2 and CXCR4 Val196^(5.35) packs near CXCL12 Val3. Deletionof residues 1-8 leads to no detectable binding, and in our model thesubstantial contribution of Arg8 results from a salt bridge to Glu32near the top of the pocket (FIG. 21B).

The site 1 interaction is characterized by the CXCL12 RFFESH (SEQ IDNO:10) motif from residues 12-17 of the N-loop region. Arg12 and His17of the RFFESH (SEQ ID NO:10) motif are stabilized in our model by Glu32and Asp181^(ECL2), respectively (FIG. 21C). One surprising feature ofour model was the relatively contiguous interaction surface. The modelwas scanned for CXCL12 residues that haven't been previously assigned toeither site 1 or 2, and would not be predictable from the current model.We identified Asn33, which is located in the CXCL12 β1-β2 loop, and ispredicted to form a potential hydrogen bond with CXCR4 Asn176^(ECL2).Mutation of Asn33 to alanine, glutamine or arginine reduced both calciumflux EC₅₀ six-fold and similarly effected chemotaxis (FIG. 21D).CXCL12:CXCR4 forms a large continuous protein-protein interface (PPI)that involves both the canonical site 1 and site 2 elements but theseinterfaces are actually part of one continuous PPI, suggesting that,despite its utility, the original two-site model is anoversimplification.

In addition to results from mutagenesis-based functional studies, wealso validated our model using experimental distance measurements. Wemapped previously published transferred cross-saturation (TCS) NMRmeasurements onto our model. The TCS experiments, performed usingfull-length CXCL12_(WT), identified CXCL12 methyl-proton resonanceswithin 5 Å of CXCR4 residues. Several methyl group resonances, includingVal18¹, Leu26⁶², Ile51 and Ile58, were excluded in the authors' originalanalyses because they were completely buried within the CXCL12 moleculeand unexplainable by the prevailing model. Not only does our modelsupport the TCS results, but it is also fully consistent with allresonances possessing intensity reductions>0.1 (FIG. 21E). Finally, thepairwise distance between residues was highly consistent with thosepreviously identified in cysteine trapping experiments, and suggest abetter overall model of the active receptor complex than previousattempts (Table 3).

TABLE 3 Pair-wise distance comparison of 1:1 CXCR4:CXCL12₁ model tocysteine trapping experiment. The efficacy of cysteine trapping fromKufareva et al. was qualitatively inspected and compared to the measureddistance in the full- length 1:1 CXCR4:CXCL12₁ model. CXCR4 CXCL12₁Cβ-Cβ (Å) Intensity Lys25 Glu15 8.6 *** Lys25 Ser16 8.9 *** Lys25 His1711.5 * Phe29 Ph13 7.4 ** Glu31 Arg8 12.7 * Glu32 Arg8 10.6 *

Comparison of CXCL12:CXCR4 Model with Previous Structures.

CXCR4 was recently crystallized in complex with vMIP-II, an antagonisticbroad-spectrum viral chemokine, by introducing a disulfide cross-linkbetween ECL2 of the receptor and the vMIP-II N-terminus. The overallcomplex geometry resembles our model with site 2 orienting the axis ofthe chemokine β-sheet roughly parallel to the transmembrane region.Whereas the CXCL12:CXCR4 site 1 interaction contributes ˜66% of thetotal binding energy, the vMIP-II:CXCR4 complex is primarily driven bythe site 2 interaction. A comprehensive comparison of the vMIP-II:CXCR4site 1 interaction with our CXCL12:CXCR4 model is not possible becausethe electron density for CXCR4 residues 1-22 is absent from the crystalstructure, presumably due to disorder. The less extensive vMIP-II site 1interface, and comparatively small contact surface (˜1330 Å² buried), isfurther consistent with previous mutagenic studies, and may also reflectits promiscuous nature.

From our model and mutagenic analyses, the CXCL12:CXCR4 site 1.5interface corresponds to CXCL12 residues 4-SLSYR-8 and should includetwo additional chemokine residues that bury ˜170 Å² of surface which isneither site 1 or 2: Arg12 (immediately after the CXC motif, firstresidue of the RFFESH (SEQ ID NO:10) motif) and Asn33 (in the middle ofthe β1-β2 loop) (FIGS. 21A-C). The vMIP-II N-loop forms anintermolecular β-sheet with CXCR4 residues Pro27-Cys28, supporting CXCR4Arg30-Ala34 to form an additional helical turn, and drawing the vMIP-IIglobular domain toward transmembrane helices I and II. The N-loop bulgetypical of CXC-type chemokines prevent similar contacts in our model,and subsequently CXCL12 is translated toward helices V and VI. Thedistinct position of the CXCL12 globular domain modifies the site 2interaction. Although the CXCL12 N-terminus is two residues shorter,Lys1 nevertheless reaches a similar depth as the vMIP-II N-terminus, andalso makes critical contacts with Asp97^(2.63) and Glu288^(7.39), butdoes not form a helical structure. The expansive site 1, 1.5, and 2interfaces in our model results in a much larger contact surface (˜3300Å²) that buries nearly 40% of the entire CXCL12 surface.

Model-Based Insight into Biased Agonism.

A tempting hypothesis for the bell-shaped chemotaxis response is CXCL12dimerization. As CXCL12₁ is incapable of self-association, but stillsignals migratory arrest, our data would suggest that ligand-receptorstoichiometry, independent of the chemokine quaternary structure, mightregulate CXCR4 function. Symmetrization of our model is compatible withtwo monomers simultaneously binding a CXCR4 dimer (FIG. 26), and it isreasonable to hypothesize that binding of chemokines in adjacent siteswould alter intracellular receptor conformation and modify signaling. Insome respects the covalent CXCL12₂ replicates the CXCR4 response to highCXCL12_(WT) or CXCL12₁ concentrations such as reduced β-arrestinrecruitment and a cellular idling phenotype. In comparison toCXCL12_(WT), the CXCL12₂ binds CXCR4₁₋₃₈ four-fold tighter butrecognizes the full-length receptor six-times weaker. Nonetheless,CXCL12₂ is a potent inhibitor of CXCL12-mediated chemotaxis. In ourmodel, unmodified substitution of CXCL12₁ with CXCL12₂ would not becompatible with the site 1 interaction in the previously determinedCXCL12₂:CXCR4₁₋₃₈ NMR structure, and would additionally result in stericclash between the CXCL12₂ helices.

Model-Based Insight into CXCR4 Inhibitors.

The pathological relevance of CXCL12:CXCR4 signaling has motivated thepursuit of small-molecule and peptide-based CXCR4 inhibitors and, morerecently, direct antagonists of CXCL12. High-throughput drug discoveryhas produced many inhibitors of site 2, including small-molecule IT1tand the CVX15 peptide inhibitor, which were recently crystallized withCXCR4. Our model illustrates how the IT1t small-molecule inhibitor couldblock CXCL12-mediated receptor activation through contacts in the minorpocket with Asp97^(2.63), Cys186^(ECL2), and Glu288^(7.39) (FIGS.21A-C). The CVX15 peptide, which is not illustrated because our modelwas generated using the IT1t model, likely blocks CXCL12 binding throughsubstantially contact overlaps to ECL2 residues Asp187-Tyr190 andhelices IV, V, and VI. A sulfotyrosine binding pocket, hypothesized tobe conserved structurally across the chemokine family, has recently beentargeted using structure-based drug design. A crystal structure ofcompound 1:CXCL12 suggests that the small-molecule would compete withCXCR4 for both hydrophobic (Val18, Leu42, and Val49) and polar contacts(Glu15, Asn22 and Arg47) on CXCL12 (FIGS. 21A-C).

In addition to molecular inhibitors, our model provides structuralhypotheses for the effects resulting from receptor and chemokinemodifications. Sulfotyrosine modification has been demonstrated toenhance ligand affinity for CCR2b, CCR5, CCR8, CXCR3, CX₃CR1, and CXCR4.One notable exception is sulfation of CXCR4 Tyr7, which is slowlysulfated in vitro and has reduced affinity for CXCL12. As illustrated inour model Tyr7 recognizes an apolar pocket formed the CXCL12 β-sheet andhelix (FIGS. 21A-C). Reactive nitrogen species produced in tumormicroenvironments were recently shown to be immunosuppressive bymodifying chemokine tyrosine residues and, subsequently, reducing theirability to recognize and activate receptors. Nitration of CXCL12 Tyr7and Tyr61 would be hypothesized to inhibit receptor interactions andcould be explained by reducing the affinity of both site 1 and 1.5(FIGS. 21A-C).

Protein Expression and Purification.

CXCR4₁₋₃₈, CXCL12_(WT), CXCL12₂, and CXCL12₁ were produced as previouslydescribed.

Radioligand Binding Competition Assay.

HEK293E cells were seeded in Dulbecco's modified Eagle's mediumsupplemented with 10% fetal bovine serum, 100 units/mLpenicillin/streptomycin (Life Technologies) in six well plates andtransiently transfected with poly-(ethylenimine) (PEI) (PolysciencesInc.) using 2 μg/well of WT, Ile4Ala/Ile6Ala, or Ile4Glu/Ile6GluFLAG-hCXCR4 cDNA vector. Radioligand binding was conducted 48 h posttransfection. Cells were washed twice in PBS (Wisent) and incubated 5minutes with 100 μM phenylarsine oxide (Sigma-Aldrich) in PBS at 37° C.Cells were washed twice and resuspended in binding buffer [50 mM HEPES,5 mM MgCl₂, 1 mM CaCl₂), 0.2% (w/v) BSA, pH 7.4], seeded in 96 well flatbottom plate at 20,000 cells per well and incubated with 50 μM¹²⁵I-CXCL12 (Perkin-ECXCL121er) as a tracer and increasingconcentrations of competing unlabelled chemokine, for 30 minutes at 37°C. Bound radioactivity was separated from free ligands by filtration onborosilicate filter paper (Molecular Devices) treated with a 0.33% PEIsolution. Receptor-bound radioactivity was quantified by gamma-radiationcounting (Perkin-ECXCL121er Life and Analytical Sciences). Bindingexperiments were carried out in duplicate.

THP-1 Calcium Response.

THP-1 monocyte cells were washed twice and resuspended in 96-well formatat 2×10⁵ cells/well in assay buffer: Hanks buffered saline solution(HBSS), 20 mM HEPES (pH 7.4), 0.1% (w/v) BSA, and FLIPR Calcium4 dye(Molecular Devices) and then incubated for 1 h at 37° C., 5% CO₂.Fluorescence was measured at 37° C. using a FlexStation3 MicroplateReader (Molecular Devices) with excitation and emission wavelengths at485 nm and 515 nm, respectively. Chemokines were resuspended at theindicated concentrations and added to the cells following a 20 sbaseline fluorescence measurement. Percent calcium flux was calculatedfrom the maximum fluorescence minus the minimum fluorescence as apercent of baseline. EC₅₀ values were determined by non-linear fittingto a four parameter logistic function.

NALM6 Chemotaxis.

NALM6 cells were grown in RPMI-1640 supplemented with 10% FBS, 100 U/mLpenicillin-streptomycin, 2 mM glutamine, 50 μM 2-mercaptoethanol,non-essential amino acids, 1 mM Na-pyruvate and 25 mM HEPES buffer (pH7.3). Chemotaxis assays were performed in triplicate in 48-well Boydenchambers (NeuroProbe), using 5 μm pore-sized polyvinylpyrrolidone-freepolycarbonate membranes. Chemotaxis medium (RPMI-1640, 25 mM HEPESsupplemented with 1% FBS) alone or chemotaxis medium containingincreasing concentrations of CXCL12 variants was added to the lowerwells. Cells (1×10⁵ per well) resuspended in chemotaxis medium wereadded to the upper well and incubated for 90 min at 37° C. in 5% CO₂atmosphere. Cells were removed from the upper part of the membrane witha rubber policeman. Cells attached to the lower side of the membranewere fixed and stained as described. Migrated cells were counted in fiverandomly selected fields of 1000-fold magnification.

MiaPaCa2 Chemotaxis.

Chemotactic migration of pancreatic cancer MiaPaCa2 cells was performedas previously described using Transwell plates coated with 15 ug/mlcollagen. Briefly, MiaPaCa2 cells were serum starved for 2 hours, liftedusing enzyme-free dissociation buffer, washed and then counted using ahemocytometer. 100,000 cells in 10 uL of serum free media were platedinto the top chamber of each Transwell. The bottom chamber of eachTranswell contained each stimulant in 500 uL serum free media. MiaPaCa2cells were allowed to migrate for 6 hours, after which the cellsremaining on the top of the chamber were swabbed out. Plates were thenfixed in 4% paraformaldehyde and stained with DAPI. Migrated cells werevisualized and counted by fluorescence microscopy, with 5 representativehigh-powered fields taken per well.

β-Arrestin-2 Recruitment.

β-arrestin-2 recruitment was measured using an intermolecular BRET assayperformed as described previously. Briefly, HEK293e cells werecotransfected with 1 μg of GFP₁₀-β-arrestin-2 construct and 0.05 μg ofCXCR4-RLuc3. All transfections were completed to 2 μg of DNA per wellwith empty vector. Following overnight culture, transiently transfectedcells were seeded in poly-D-lysine-coated 96-well white clear-bottommicroplates (View-Plate, Perkin-ECXCL12₁er Life and Analytical Sciences)and left in culture for 24 h. The medium of the cells was then changedto BRET buffer (PBS, 0.5 mM MgCl₂, 0.1% (w/v) BSA). 0-arrestin-2recruitment was measured 15 min after ligand addition, and 10 min afterthe addition of the RLuc3 substrate coelenterazine400a (NanoLightTechnology) at a final concentration of 5 mM. The values were correctedto BRETnet by subtracting the background BRET signal obtained in cellstransfected with the luciferase construct alone.

Agarose Microdroplet Assay.

The agarose microdroplet assay was performed to determine U-937 cellularmigration, as previously described {AdeCXCL12₁an, 1980 #1609}. 0.5×10⁶cells/ml U-937 target cells were harvested and washed in HBSS. Cellswere spun at 2,000 rpm and were transferred to a graduated 15 ml glassconical tube. The cell concentration was adjusted in agarose medium,prepared from 2% low melting temperature Seaplaque agarose and mediumcontaining 15% FBS, using a 1:4 volume-to-volume dilution. A 1 agarosedroplet containing 1×10⁵ target cells was placed in the center of eachwell of a 96-well flat-bottom tissue culture plate, in triplicate, usinga gastight 0.05 ml Hamilton syringe (Hamilton Company, Reno, Nev.).Droplets were allowed to harden at 4° C. for 20 min. 200 chilled testmedia was applied to each well. Test medium consisted of serum-freemedium, 25% FBS, and the indicated CXCL12 concentrations. The plate wasincubated for 18-24 h at 37° C. and 5% CO₂. Following incubation, theradius of each droplet was determined and target cell migration wasmeasured at four directional points 900 from one another, using aninverted light microscope equipped with a gridded eyepiece, at 40× totalmagnification. Percent inhibition of each sample was quantified. Theplate was incubated for an additional 24 h to measure recovery, andviability was determined by trypan blue exclusion.

NMR Structure Determination.

All NMR spectra were acquired on a Bruker DRX 600 MHz spectrometerequipped with a ¹H, ¹⁵N, ¹³C TXI cryoprobe at 298 K. Experiments wereperformed in a solution containing 25 mM deuterated MES (pH 6.8), 10%(v/v) D₂O, and 0.02% (w/v) NaN₃. NOE distance restraints were obtainedfrom 3D ¹⁵N-edited NOESY-HSQC, aliphatic ³C-edited NOESY-HSQC, andaromatic ³C-edited NOESY-HSQC spectra (τ_(mix)=80 ms) collected on both[U-¹⁵N,¹³C]-CXCL12₁ saturated with CXCR4₁₋₃₈ and [U-¹⁵N,¹³C]-CXCR₁₋₃₈saturated with CXCL12₁. Intermolecular NOEs were obtained from a 3DF1-¹³C/¹⁵N-filtered/F3-¹³C-edited NOESY-HSQC (τ_(mix)=120 ms) collectedon both [U-¹⁵N,¹³C]-CXCL12₁ saturated with CXCR4₁₋₃₈ and[U-¹⁵N,¹³C]-CXCR₁₋₃₈ saturated with CXCL12₁. In addition to NOEs,backbone φ/ψ dihedral angle restraints were derived from ¹H^(N), ¹H^(α),¹³C^(α), ¹³C^(β), ¹³C′ and ¹⁵N chemical shift data using TALOS+. Bothdistance and dihedral restraints were used to generate initial NOEassignments and preliminary structures via the NOEASSIGN module ofCYANA. Complete structure determination was undertaken as an iterativeprocess of correcting/assigning NOEs and running structure calculationswith CYANA. The 20 CYANA conformers with the lowest target function werefurther refined by a molecular dynamics protocol in explicit solventusing XPLOR-NIH.

2D NMR Characterization.

All NMR spectra were acquired on a Bruker DRX 600 MHz spectrometerequipped with ¹H, ¹⁵N, ¹³C TXI cryoprobe at 298 K. Experiments wereperformed in a solution containing 25 mM deuterated MES (pH 6.8), 10%(v/v) D₂O, and 0.02% (w/v) NaN₃. Heteronuclear NOE experiments werecollected on 250 μM [U-¹⁵N]-CXCR4₁₋₃₈ in the absence and presence of 500μM CXCL12₁. ¹⁵N-HSQC spectra were collected to monitor the interactionof 200 μM [U-¹⁵N]-CXCR4₁₋₃₈ was titrated with 0, 100, 200, 300, 400, and500 μM CXCL12₂. ¹⁵N-HSQC spectra were collected to monitor theinteraction of 250 μM [U-¹⁵N]-CXCR4₁₋₃₈ was titrated with 0, 62.5, 125,187.5, 250, 375 and 500 μM CXCL12₁.

Calcium Response of CXCR4 Mutants.

Cell culture, transfection and calcium flux assay of Chinese hamsterovary K1 (CHO-K1) cells was performed as previously described

Cell Lines, Antibodies and Other Reagents.

HEK (Human embryonic kidney) 293 (Microbix, Toronto, Canada) and HeLa(American Type Culture Collection) cells were maintained in Dulbecco'smodified Eagles medium (DMEM; Hyclone) supplemented with 10% fetalbovine serum (FBS; HyClone Laboratories, Logan, Utah). The PE-conjugatedCXCR4 (CD184) isotype control antibodies, and rat anti-CXCR4 (2B11) andwere from BD Biosciences (San Jose, Calif.). The anti-actin antibody wasfrom MP Biomedicals (Aurora, Ohio). The Alexa-Fluor 568-conjugated goatanti-rabbit antibody was from Molecular Probes (Eugene, Oreg.). Theanti-FLAG M2 horseradish peroxidase conjugated monoclonal antibody wasfrom Sigma (St. Louis, Mo.). The anti-HA polyclonal and monoclonalantibodies were from Covance (Berkeley, Calif.). CXCL12 was fromPeproTech (Rockyhill, N.J.).

CXCR4 Surface Expression.

In parallel to radioligand binding, the expression of CXCR4 variants atthe cell surface of the transfected cells was assessed using flowcytometry. 48 h after transfection, 1×10⁶ cells were collected andwashed twice in cold PBS. Cells were then stained withCXCR4-phycoerythrin (clone 12G5; eBioscience) or isotype-phycoerythrinantibody according to the manufacturer's recommendation. Thefluorescence was measured using a FACSCalibur flow cytometer (BDBioscience).

CXCR4 Degradation Assay.

HeLa cells grown on 6-cm dishes were washed once and incubated with DMEMcontaining 10% FBS and 50 μg/ml cyclohexamide for 15 min at 37° C. Cellswere then incubated in the same medium containing vehicle (PBScontaining 0.5% bovine serum albumin [BSA]), 80 ng/ml CXCL12_(WT),CXCL12₁, or LD for 3 h. Cells were washed once and collected in 300 μl2× sample buffer. Receptor levels were determined by SDS-PAGE followedby immunoblotting using an anti-CXCR4 antibody. Blots were stripped andreprobed for actin to assess loading. Receptor levels normalized toactin levels were determined by densitometric analysis.

CXCR4 Internalization Assay.

HeLa cells grown on 6-cm dishes were washed twice with PBS and detachedfrom the surface of the plate by incubating with 350 μl Cellstrippercell dissociation solution (Mediatech, VA) for 10 min at 37° C. Cellswere collected in 4 ml PBS containing 0.1% BSA (Media Tech, VA) bycentrifugation. Cell pellets were re-suspended in 1.5 ml PBS-0.1% BSAand 5×10⁵ cells were transferred to a fresh 5 ml polystyrene roundbottom tube (BD falcon) and washed once with PBS+0.1% BSA andre-suspended in 250 μl PBS-0.1% BSA. Cells were incubated at 37° C. for15 min and then treated with vehicle, 80 ng/ml CXCL12_(WT), CXCL12₁, orLD for 20 min at 37° C. Following treatment, 4 mL cold PBS was added toeach tube, cells were collected by centrifugation and then fixed byre-suspending the pellet in 500 μl 4% paraformaldehyde (made in PBS) andincubating for 15 min at 37° C. Cells were collected by centrifugationand washed once with 4 mL PBS and then twice with PBS-0.1% BSA. Cellswere stained with PE-conjugated anti-CXCR4 or isotype control antibodiesby re-suspending the pellet in 100 μl PBS-0.1% BSA (supplemented with 5%normal goat serum) containing anti-CXCR4 antibody (1:100 dilution) andincubating for 1 hour at room temperature in the dark. Followingstaining, cells were washed twice with 4 mL PBS-0.1% BSA. Finally, cellswere re-suspended in 300 μl PBS-0.1% BSA and kept in the dark until theanalysis was performed. CXCR4 surface expression was analyzed by flowcytometry (FACS-CANTO; Becton Dickinson) and analysis was performedusing FlowJo v.9.3 software.

CXCR4 Ubiquitination Assays.

HEK293 cells stably expressing HA-CXCR4 grown on 10-cm dishes weretransfected with 3 μg FLAG-ubiquitin. The next day, cells were passagedonto 6-cm dishes and allowed to grow for an additional 24 h. Thefollowing day, cells were incubated in DMEM containing 20 mM HEPES for 6h and then treated with vehicle (PBS, 0.1% BSA), 80 ng/ml CXCL12_(WT),CXCL12₁, or LD for 30 min. Cells were then washed once on ice with coldPBS, and collected in 1 ml lysis buffer [50 mM Tris-Cl, pH 7.4, 150 mMNaCl, 5 mM EDTA, 0.5% (w/v) sodium deoxycholate, 1% (v/v) NP-40, 0.1%(w/v) SDS, 20 mM N-ethyCXCL12₁aleimide (NEM), and 10 μg/ml each ofleupeptin, aprotinin, and pepstatin A]. Samples were transferred intomicrocentrifuge tubes and placed at 4° C. for 30 min, followed bysonification and centrifugation to pellet cellular debris. Clarifiedcell lysates were incubated with an anti-HA polyclonal and isotypecontrol antibodies and the immunoprecipitates were analyzed by SDS-PAGEfollowed by immunoblotting using an anti-FLAG antibody conjugated toHRP.

Confocal Microscopy.

HeLa cells transiently transfected with HA-CXCR4-YFP were passaged ontopoly-L-lysine coated coverslips and grown for 24 h. The next day, cellswere washed once with warm DMEM containing 20 mM HEPES, pH 7.5, andincubated in the same medium for 3-4 h at 37° C. Cells were treated with80 ng/ml CXCL12_(WT), CXCL12₁, or LD and vehicle for 30 min. Cells werethen fixed with PBS containing 3.7% paraformaldehyde, followed bypermeabilization with 0.05% (w/v) saponin for 10 min, similar to aprotocol we have described previously. Cells were then incubated with 1%goat serum in 0.05% saponin-PBS for 30 min at 37° C., followed bystaining with anti-mouse monoclonal antibody that recognizes duallyphosphorylated CXCR4 on serine residues 324 and 325 (clone 5E11) (1:50dilution) for 1 h at 37° C. Cells were washed five times with 0.05%saponin-PBS, followed by incubating with Alexa-Fluor 568-conjugatedanti-rabbit antibody for 30 min at 37° C. (1:200 dilution). Finally,cells were washed five times with 0.05% saponin-PBS and mounted ontoglass slides using mounting media containing4,6-diamidino-2-phenylindole (Vectashield mounting media with DAPI).Samples were analyzed using an LSM 510 laser scanning confocalmicroscope (Carl Zeiss, Thornwood, N.Y.) equipped with a Plan-Apo63×/1.4 oil lens objective. Images were acquired using a 1.4-megapixelcooled extended spectra range RGB digital camera set at 512×512resolution. Acquired images were analyzed using ImageJ, version 1.41osoftware (National Institutes of Health, Bethesda, Md.) and AdobePhotoshop (CS4).

Apoptosis Assays.

KG1a (human acute myelogenous leukemia) cells were assayed forCXCL12-dependent apoptosis as previously described. Briefly, CXCR4expression on KG1a cells was achieved via transient transfection with aplasmid encoding a CXCR4-YFP fluorescent fusion protein. These cellswere treated with the indicated concentration of each CXCL12 variant andcultured for 16-18 h prior to measuring apoptosis. Apoptosis was assayedby staining cells with APC-conjugated annexin V to detect cell-surfacephosphatidyl serine (BD Biosciences) or Alexaflour-647-conjugatedantibody to detect cleaved PARP (BD Biosciences), and the percentage ofYFP+ cells undergoing apoptosis was determined by flow cytometry.

Modeling CXCR4:CXCL12₁ Complex—Docking of CXCL12₁ N-Terminal Peptide toCXCR4.

The CXCL12₁ N′ peptide (residues 1-8; KPVSLSYR) was docked into theorthosteric site of CXCR4 (PDB 30DU:A residues 29-301). The peptide wasmanually placed away from the receptor, at a canonical extendedconformation ((φ,ψ+−135). 90,000 models were generated using FlexPepDockAb-Initio, out of which the top 500 by reweighted_sc were clustered (ata threshold of 2 Å peptide RMSD). Next, four representative clustercenters in which the peptide was occupying the presumed binding sitewere selected to seed the generation of additional 100,000 models, againusing FlexPepDock Ab-Initio, and the top 500 were clustered as before. Atop scoring cluster representative was selected based on electrostaticinteractions with experimentally important CXCR4 residues (D97/E288).

Initial Placement of SDF C-Terminal Domain.

A truncated version of the CXCL12₁/CXCR4₁₋₃₈ NMR complex (CXCL12₁residues 9-78, CXCR4₁₋₃₈ residues 4-26; both taken from the first modelof the NMR ensemble) was manually placed onto CXCR4, such that CXCL12₁Cys9 was in the vicinity of Arg8 of the docked N′ peptide from step 1,and Pro27 of CXCR4 will be in the vicinity of Glu26 (from theCXCL12₁/CXCR4₁₋₃₈ NMR complex). We considered Pro27 to be relativelyrigid as it is followed by Cys28, which forms a disulfide bridge toCys274. To validate this initial manual placement, a local RosettaDockrun was initiated from this starting pose and indeed resulted in anoticeable energy funnel leading towards the starting pose.

Computational Relaxation of the Complex Between CXCL12₁ and CXCR4.

The manually placed CXCL12₁/CXCR4 complex from step 2 was subjected to aRosetta Relax run, generating 5,000 models in which the backbone andsidechain conformations of the protein partners and the rigid bodyorientation were optimized. The top model according to Rosetta Score12was progressed to loop modeling.

Loop Modeling Between CXCR4₁₋₃₈ and CXCR4.

Rosetta loop modeling protocol was used to model CXCR4 residues Tyr21 toPro27 and close the loop between the CXCR4₁₋₃₈ fragment from the NMRmodel (residues 4-26) and CXCR4 X-ray structure (residues 29-301). 1000loop models were generated in the presence of CXCL12₁ using theperturb/refine kinematic loop closure protocol. The top scoring modelwas used for the subsequent modeling step.

Redocking of CXCL12₁ N-Terminal Peptide and CXCL12₁ Loop Closure.

Using FlexPepDock Ab-Initio as in step 1, we produced 30,000 models ofCXCL12₁ N′ peptide (residues 1-8) starting from its previous pose (seeabove), but this time using constraints that would later forceconnectivity to residue Cys9 of CXCL12₁ coordinates from step 3 (BoundRosetta restraints with standard deviation of 0.1 Å), and contact withkey binding site residue Asp97 and Glu288 (distance 1.25-1.35 Å for Catom of residue 8 and N atom of residue 9; distance 2.35-2.45 Å for Catom of residue 8 and CA atom of residue 9; distance 2.35-2.45 Å for CAatom of residue 8 and N atom of residue 9; Binding site constraints onAsp97 and Glu288, in both centroid mode (range 1.0-5.5 Å for Asp97 CBatom and 1.0-6.5 Å for Glu288 CB atom), and in full atom mode (range1.0-5.0 Å for both Asp97 CB atom and Glu288 CG atom).

The top 500 models were clustered as previously and cluster #27 wasselected from the top scoring clusters as a representative favorablecontacts to previously defined binding site residues. A second round ofpeptide docking was performed starting from this model now in thepresence of the CXCL12₁/CXCR4₁₋₃₈ complex (except for Cys9 that wasomitted to avoid clashes). The same constraints were used, to keep thepeptide close to Asp97/Glu288 and to force connectivity to Cys9 in nextstep. The top scoring model was used for loop closure of residues 6-9(as above) to connect CXCL12₁ N′ peptide to rest of CXCL12₁. Finallyanother relaxation of the complete complex generated 100 models in whichcorrect disulfide topology was enforced. The top scoring model of thisrelax run is presented.

Statistical Analysis and Final Figures.

Data were analyzed by one-way analysis of variance (ANOVA) usingGraphPad Prism 4.0 (GraphPad Software).

While this invention has been described in conjunction with the variousexemplary embodiments outlined above, various alternatives,modifications, variations, improvements and/or substantial equivalents,whether known or that are or may be presently unforeseen, may becomeapparent to those having at least ordinary skill in the art.Accordingly, the exemplary embodiments according to this invention, asset forth above, are intended to be illustrative, not limiting. Variouschanges may be made without departing from the spirit and scope of theinvention. Therefore, the invention is intended to embrace all known orlater-developed alternatives, modifications, variations, improvements,and/or substantial equivalents of these exemplary embodiments. Alltechnical publications, patents and published patent applications citedherein are hereby incorporated by reference in their entirety for allpurposes.

REFERENCES CITED

-   1. Ma et al. (1998) Proc. Natl. Acad. Sci. U.S.A 95, 9448-9453.-   2. Zou et al. (1998) Nature 393, 595-599.-   3. Sierro et al. (2007) Proc. Natl. Acad. Sci. U.S.A 104,    14759-14764.-   4. Burns et al. (2006) J. Exp. Med. 203, 2201-2213.-   5. Hu et al. (2007) Circulation 116, 654-663.-   6. Saxena et al. (2008) Circulation 117, 2224-2231.-   7. Proulx et al. (2007) Pfluegers Arch. 455, 241-250.-   8. Endres et al. (1996) Cell 87, 745-756.-   9. Balkwill, F. (2004) Nat. Rev. Cancer 4, 540-550.-   10. Crump et al. (1997) EMBO J. 16, 6996-7007.-   11. Kofuku et al. (2009) J. Biol. Chem. 284, 35240-35250.-   12. Farzan et al. (2002) J. Biol. Chem. 277, 29484-29489.-   13. Seibert et al. (2008) Biochemistry 47, 11251-11262.-   14. Farzan et al. (1999) Cell 96, 667-676.-   15. Preobrazhensky et al. (2000) J. Immunol. 165, 5295-5303.-   16. Bannert et al. (2001) J. Exp. Med. 194, 1661-1673.-   17. Colvin et al. (2006)Mol. Cell. Biol. 26, 5838-5849.-   18. Gutierrez et al. (2004) J. Biol. Chem. 279, 14726-14733.-   19. Fong et al. (2002) J. Biol. Chem. 277, 19418-19423.-   20. Rajarathnam et al. (1994) Science 264, 90-92.-   21. Paavola et al. (1998) J. Biol. Chem. 273, 33157-33165.-   22. Laurence et al. (2000) Biochemistry 39, 3401-3409.-   23. Proudfoot et al. (2003) Proc. Natl. Acad. Sci. U.S.A 100,    1885-1890.-   24. Tan et al. (2012) J. Biol. Chem. 287, 14692-14702.-   25. Veldkamp et al. (2008) Sci. Signaling 1, ra4.-   26. Drury et al. (2011) Proc. Natl. Acad. Sci. U.S.A-   27. Takekoshi et al. (2012) Mol. Cancer Ther. 11, 2516-2525.-   28. Veldkamp et al. (2006) J. Mol. Biol. 359, 1400-1409.-   29. Veldkamp et al. (2010) J. Am. Chem. Soc. 132, 7242-7243.-   30. Ziarek et al. (2013) Curr. Top. Med. Chem. 12, 2727-2740.-   31. Ziarek et al. (2011) Int. J. Mol. Sci. 12, 3740-3756.-   32. Veldkamp et al. (2005) Protein Sci. 14, 1071-1081.-   33. Veldkamp et al. (2009) Protein Sci. 18, 1359-1369.-   34. Dombkowski, A. A. (2003) Bioinformatics 19, 1852-1853.-   35. Gozansky et al. (2005) J. Mol. Biol. 345, 651-658.-   36. Simpson et al. (2009) Chem. Biol. 16, 153-161.-   37. Zhu et al. (2011) Biochemistry 50, 1524-1534.-   38. Tan et al. (2013) J. Biol. Chem. 288, 10024-10034.-   39. Wells, J. A., and McClendon, C. L. (2007) Nature 450, 1001-1009.-   40. Nielsen et al. (2012) Haemophilia 18, e397-398.-   41. Duma et al. (2007) J. Mol. Biol. 365, 1063-1075.-   42. Pawson, T., and Nash, P. (2003) Science 300, 445-452.-   43. Edwards et al. (2007) PLoS ONE 2, e967.-   44. Machida, K., and Mayer, B. J. (2005) Biochim. Biophys. Acta    1747, 1-25.-   45. Vidler et al. (2012) J. Med. Chem. 55, 7346-7359.-   46. Herold et al. (2011) Curr. Chem. Genomics 5, 51-61.-   47. Stone et al. (2009) New Biotechnol. 25, 299-317.-   48. Peterson, F. C., and Volkman, B. F. (2009) Front. Biosci. 14,    833-846.-   49. Monigatti et al. (2002) Bioinformatics 18, 769-770.-   50. Liu et al. (2008) Am. J. Respir. Cell Mol. Biol. 38, 738-743.-   51. Keller, R. (2004) The Computer Aided Resonance    Assignment/Tutorial, CANTINA, Zurich.-   52. Ziarek, et al. (2011) Methods Enzymol. 493, 241-275.

1. A method of treating an autoimmune disease in a subject in needthereof, the method comprising administering to the subject atherapeutically effective amount of a composition comprising aconstitutively monomeric CXCL12 peptide comprising the amino acidsequence of SEQ ID NO:1 wherein the amino acids at positions 55 and 58are substituted with cysteine.
 2. The method of claim 1, wherein CXCL12peptide is CXCL12₁ having the amino acid sequence of SEQ ID NO:2.
 3. Themethod of claim 1, wherein the autoimmune disease is selected from thegroup consisting of Multiple Sclerosis (MS), Guillain-Barre Syndrome,Amyotrophic Lateral Sclerosis, Parkinson's disease, Alzheimer's disease,Type I diabetes, lupus, ankylosing spondylitis, rheumatoid arthritis,psoriatic arthritis, juvenile arthritis, early arthritis, reactivearthritis, osteoarthritis, ankylosing spondylitis, autoimmune uveitis,and inflammatory bowel diseases.
 4. The method of claim 1, wherein theautoimmune disease is Type I diabetes.
 5. The method of claim 1, whereinthe subject is a mammal.
 6. The method of claim 4, wherein the subjectis a human.
 7. The method of claim 1, wherein the composition furthercomprises a pharmaceutically acceptable carrier or diluent.